FIELD OF THE INVENTION The present invention relates to SEM inspection of fluid containing samples generally and more particularly to sample containers and inspection systems as well as methods for utilization thereof.

BACKGROUND OF THE INVENTION The following U. S. patent documents are believed to represent the current state of the art: 4,071, 766; 4,720, 633; 5,250, 808; 5,326, 971; 5,362, 964; 5,412, 211; 4,705, 949; 5,945, 672; 6,365, 898; 6,130, 434; 6,025, 592; 5,103, 102; 4,596, 928; 4,880, 976; 4,992, 662; 4,720, 622; 5,406, 087; 3,218, 459; 3,378, 684; 4,037, 109; 4,448, 311; 4,115, 689; 4,587, 666; 5,323, 441; 5,811, 803; 6,452, 177; 5,898, 261; 4,618, 938; 6,072, 178; 6,114, 695 and 4, 929,041.

SUMMARY OF THE INVENTION The present invention seeks to provide apparatus, systems and methodologies for enabling SEM inspection of fluid containing samples.

There is thus provided in accordance with a preferred embodiment of the present invention a SEM compatible sample container including a sample enclosure including an electron beam permeable, fluid impermeable membrane, and a peripheral enclosure sealed to the membrane and defining with the membrane the sample enclosure, and a sample enclosure closure including quick-connect attachment functionality for sealing engagement with the sample enclosure.

In accordance with another preferred embodiment of the present invention the quick-connect attachment functionality includes a threaded connection.

Preferably, the peripheral enclosure is at least partially electrically conductive.

Additionally, the SEM compatible sample container also includes a pressure relief diaphragm associated with the sample enclosure. Additionally or alternatively the SEM compatible sample container also includes at least one membrane support grid supporting the membrane.

In accordance with another preferred embodiment of the present invention the membrane is formed from a material selected from the group consisting of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, PARLODION, COLLODION, KAPTON, FORMVAR, VINYLEC, BUTVAR, PIOLOFORM, PARYLENE, silicon dioxide, silicon monoxide and carbon. Preferably, the sample enclosure is preassembled and ready to receive a liquid containing sample therein, following which the sample enclosure closure may be readily sealingly joined thereto by means of the quick-connect attachment functionality.

There is also provided in accordance with another preferred embodiment of the present invention a SEM compatible liquid sample container including a liquid sample enclosure including an electron beam permeable, fluid impermeable membrane, and a peripheral enclosure sealed to the membrane and defining with the membrane the liquid sample enclosure capable of containing a liquid at a depth which is not permeable by electrons having an energy level of less than 50KeV.

In accordance with another preferred embodiment of the present invention the SEM compatible liquid sample container also includes a liquid sample enclosure closure including quick-connect attachment functionality for sealing engagement with the liquid sample enclosure. Preferably, the quick-connect attachment functionality includes a threaded connection. Additionally, the peripheral enclosure is at least partially electrically conductive. Alternatively or additionally, the SEM compatible liquid sample container also includes a pressure relief diaphragm associated with the liquid sample enclosure.

In accordance with yet another preferred embodiment of the present invention the SEM compatible liquid sample container also includes at least one membrane support grid supporting the membrane. Preferably, the membrane is formed from a material selected from the group consisting of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, PARLODION, COLLODION, KAPTON, FORMVAR, VINYLEC, BUTVAR, PIOLOFORM, PARYLENE, silicon dioxide, silicon monoxide and carbon. Additionally, the sample enclosure is preassembled and ready to receive a liquid containing sample therein, following which the sample enclosure closure may be readily sealingly joined thereto by means of the quick-connect attachment functionality.

There is further provided in accordance with yet another preferred embodiment of the present invention a SEM compatible sample container including a sample dish including an electron beam permeable, fluid impermeable, membrane, and a peripheral enclosure sealed to the membrane and defining with the membrane the sample dish, and an outer enclosure arranged about the sample dish and defining an aperture for electron communication through the membrane with the interior of the dish.

In accordance with another preferred embodiment of the present invention the sample dish is capable of containing a liquid at a depth which is not permeable by electrons having an energy level of less than 50KeV. Preferably, the SEM compatible sample container also includes an outer enclosure closure including quick-connect attachment functionality for sealing engagement with the peripheral enclosure. Additionally, the quick-connect attachment functionality includes a threaded connection. Additionally or alternatively, the peripheral enclosure is at least partially electrically conductive.

In accordance with yet another preferred embodiment of the present invention the SEM compatible sample also includes a pressure relief diaphragm associated with the sample dish. Preferably, the SEM compatible sample container also includes at least one membrane support grid supporting the membrane. Additionally, the membrane is formed from a material selected from the group consisting of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, PARLODION, COLLODION, KAPTON, FORMVAR, VINYLEC, BUTVAR, PIOLOFORM, PARYLENE, silicon dioxide, silicon monoxide and carbon. Alternatively or additionally, the sample enclosure is preassembled and ready to receive a liquid containing sample therein, following which the outer enclosure closure may be readily sealingly joined thereto by means of the quick-connect attachment functionality.

There is also provided in accordance with yet another preferred embodiment of the present invention a SEM compatible sample container including an enclosure defining an aperture for electron communication, and a sample dish located at the interior of the enclosure and including an electron beam permeable, fluid impermeable, membrane, the aperture being arranged with respect to the membrane for electron communication with the interior of the enclosure through the membrane.

In accordance with another preferred embodiment of the present invention the sample dish is defined by the membrane together with the enclosure.

Preferably, the sample dish is defined by the membrane together with a separate dish wall disposed within the enclosure. Additionally, the separate dish wall is sealed to the membrane. Alternatively or additionally, the sample dish is capable of containing a liquid at a depth which is not permeable by electrons having an energy level of less than 50KeV.

In accordance with yet another preferred embodiment of the present invention the SEM compatible sample container also includes a closure including quick-connect attachment functionality for sealing engagement with the enclosure.

Preferably, the quick-connect attachment functionality includes a threaded connection.

Additionally, the enclosure is at least partially electrically conductive. Alternatively or additionally, the SEM compatible sample container also includes a pressure relief diaphragm associated with the sample dish.

In accordance with still another preferred embodiment of the present invention the SEM compatible sample container also includes at least one membrane support grid supporting the membrane. Preferably, the membrane is formed from a material selected from the group consisting of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, PARLODION, COLLODION, KAPTON, FORMVAR, VINYLEC, BUTVAR, PIOLOFORM, PARYLENE, silicon dioxide, silicon monoxide and carbon. Additionally, the sample enclosure is preassembled and ready to receive a liquid containing sample therein, following which the closure may be readily sealingly joined thereto by means of the quick-connect attachment functionality.

There is further provided in accordance with yet another preferred embodiment of the present invention a SEM compatible sample container including a sample dish assembly defining an aperture for electron communication therethrough, the sample dish assembly includes an electron beam permeable, fluid impermeable, membrane which at least partially defines a sample enclosure, and a sample positioner arranged to position a sample adjacent to the membrane, the aperture being arranged with respect to the membrane for electron communication therethrough and through the membrane, with the sample adjacent thereto.

In accordance with another preferred embodiment of the present invention the SEM compatible sample container also includes a closure including quick-connect attachment functionality for sealing engagement with the enclosure.

Preferably, the quick-connect attachment functionality includes a threaded connection.

Additionally, the sample enclosure is at least partially electrically conductive.

Preferably, the sample positioner includes a spring.

In accordance with yet another preferred embodiment of the present invention the SEM compatible sample container also includes a pressure relief diaphragm associated with the sample dish assembly. Preferably, the SEM compatible sample container also includes at least one membrane support grid supporting the membrane. Additionally, the membrane is formed from a material selected from the group consisting of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, PARLODION, COLLODION, KAPTON, FORMVAR, VINYLEC, BUTVAR, PIOLOFORM, PARYLENE, silicon dioxide, silicon monoxide and carbon.

Alternatively or additionally, the sample enclosure is preassembled and ready to receive

a sample therein, following which the closure may be readily sealingly joined thereto by means of the quick-connect attachment functionality.

There is further provided in accordance with still another preferred embodiment of the present invention a SEM compatible sample container including a sample dish assembly defining an aperture for electron communication therethrough, the sample dish assembly includes an electron beam permeable, fluid impermeable, membrane which at least partially defines a sample enclosure, and a light guide arranged to receive light from a sample in the sample enclosure during SEM inspection, the light guide being arranged with respect to the sample enclosure for collecting light from the sample.

In accordance with another preferred embodiment of the present invention the light guide receives light from a side of the sample not facing the membrane. Preferably the sample dish assembly is capable of containing a liquid at a depth which is not permeable by electrons having an energy level of less than 50KeV.

Additionally, light guide is arranged to physically contact the liquid. Alternatively or additionally, the SEM compatible sample container also includes a closure including quick-connect attachment functionality for sealing engagement with the sample enclosure.

In accordance with yet another preferred embodiment of the present invention the quick-connect attachment functionality includes a threaded connection.

Preferably, the sample enclosure is at least partially electrically conductive.

Additionally, the SEM compatible sample container also includes a pressure relief diaphragm associated with the sample dish assembly. Alternatively or additionally, the SEM compatible sample container also includes at least one membrane support grid supporting the membrane.

In accordance with still another preferred embodiment of the present invention the membrane is formed from a material selected from the group consisting of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, PARLODION, COLLODION, KAPTON, FORMVAR, VINYLEC, BUTVAR, PIOLOFORM, PARYLENE, silicon dioxide, silicon monoxide and carbon. Preferably, the sample enclosure is preassembled and ready to receive a liquid containing sample therein,

following which closure may be readily sealingly joined thereto by means of the quick-connect attachment functionality.

There is also provided in accordance with another preferred embodiment of the present invention a SEM compatible sample container including a sample dish assembly defining an aperture for electron communication therethrough, the sample dish assembly includes an electron beam permeable, fluid impermeable, membrane which at least partially defines a sample enclosure, and a pressure relief diaphragm associated with the sample dish assembly. Preferably, the pressure relief diaphragm is located within the sample enclosure. Additionally, the SEM compatible sample container also includes a closure including quick-connect attachment functionality for sealing engagement with the sample enclosure. Alternatively or additionally the quick-connect attachment functionality includes a threaded connection.

In accordance with another preferred embodiment of the present invention the sample enclosure is at least partially electrically conductive. Preferably, the SEM compatible sample container also includes at least one membrane support grid supporting the membrane. Additionally, the membrane is formed from a material selected from the group consisting of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, PARLODION, COLLODION, KAPTON, FORMVAR, VINYLEC, BUTVAR, PIOLOFORM, PARYLENE, silicon dioxide, silicon monoxide and carbon. Preferably, the sample enclosure is preassembled and ready to receive a liquid containing sample therein, following which the closure may be readily sealingly joined thereto by means of the quick-connect attachment functionality.

There is further provided in accordance with yet another preferred embodiment of the present invention a SEM compatible sample container including a sample dish assembly defining an aperture for electron communication therethrough, the sample dish assembly includes an electron beam permeable, fluid impermeable membrane which at least partially defines a sample enclosure, and at least one membrane support grid supporting the membrane. Preferably, the SEM compatible sample container also includes a closure including quick-connect attachment functionality for sealing engagement with the sample enclosure. Additionally, the quick-connect attachment functionality includes a threaded connection. Alternatively or additionally, the sample enclosure is at least partially electrically conductive.

In accordance with another preferred embodiment of the present invention the SEM compatible sample container also includes a pressure relief diaphragm associated with the sample dish assembly. Preferably, the membrane is formed from a material selected from the group consisting of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, PARLODION, COLLODION, KAPTON, FORMVAR, VINYLEC, BUTVAR, PIOLOFORM, PARYLENE, silicon dioxide, silicon monoxide and carbon. Additionally, the sample enclosure is preassembled and ready to receive a liquid containing sample therein, following which closure may be readily sealingly joined thereto by means of the quick-connect attachment functionality.

There is further provided in accordance with still another preferred embodiment of the present invention a SEM compatible multiple sample container including an enclosure defining a multiplicity of apertures arranged in an array for electron communication therethrough, and at least one electron beam permeable, fluid impermeable membrane disposed over the multiplicity of apertures, the enclosure and the at least one membrane defining a multiplicity of sample dishes, each including at least a portion of the at least one membrane, and the multiplicity of apertures being arranged with respect to the at least one membrane for electron communication through the at least one membrane with interiors of the sample dishes.

Preferably, the SEM compatible multiple sample container also includes an enclosure cover assembly operative for selective individual sealing of each of the multiplicity of sample dishes. Additionally, each of the multiplicity of sample dishes is defined by the membrane together with a separate dish wall definer disposed within the enclosure. Alternatively or additionally, the separate dish wall definer is sealed to at least a portion of the at least one membrane. Additionally, the SEM compatible multiple sample container is dimensioned so as to be compatible with conventional cell biology equipment.

There is also provided in accordance with yet another preferred embodiment of the present invention a SEM compatible premicroscopy multiple sample container system including a plurality of SEM compatible sample containers, and a support for supporting the plurality of SEM compatible sample containers, wherein the membrane defines a lower surface of a sample receiving volume. Preferably, the support includes a light transparent portion underlying at least one of the membranes, whereby

light microscopy may be carried out on samples in at least one of the plurality of SEM compatible sample containers while they are supported in the support. Additionally, the SEM compatible premicroscopy multiple sample container system also includes a cover arranged to enclose the support and the plurality of SEM compatible sample containers supported thereon. Alternatively or additionally, the support includes at least one liquid reservoir for holding liquid useful in maintaining humidity of the samples in the plurality of SEM compatible sample containers while they are supported in the support.

In accordance with yet another preferred embodiment of the present invention the SEM compatible multiple sample container is provided with a suction device and pipettes. Preferably, the pipettes are provided with collar elements to prevent inadvertent engagement of the pipettes with the membrane. Additionally, the premicroscopy multiple sample container is dimensioned so as to be compatible with conventional cell biology equipment.

There is further provided in accordance with another preferred embodiment of the present invention a SEM system including a SEM, a sample dish assembly defining an aperture for electron communication therethrough, the sample dish assembly including an electron beam permeable, fluid impermeable, membrane which at least partially defines a sample enclosure, and an X-ray detector arranged to receive X-rays from a sample containing liquid located in the sample enclosure during SEM inspection. Preferably, the SEM system also includes a sample enclosure closure including quick-connect attachment functionality for sealing engagement with the sample enclosure. Additionally, the quick-connect attachment functionality includes a threaded connection.

In accordance with another preferred embodiment of the present invention the sample enclosure is at least partially electrically conductive. Preferably, the SEM system also including a pressure relief diaphragm associated with the sample enclosure. Additionally, the SEM system also including at least one membrane support grid supporting the membrane. Alternatively or additionally, the membrane is formed from a material selected from the group consisting of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, PARLODION, COLLODION, KAPTON, FORMVAR, VINYLEC, BUTVAR, PIOLOFORM, PARYLENE, silicon dioxide, silicon monoxide and carbon. Preferably, the sample enclosure is preassembled and

ready to receive a liquid containing sample therein, following which the sample enclosure closure may be readily sealingly joined thereto by means of the quick-connect attachment functionality.

There is further provided in accordance with still another preferred embodiment of the present invention a SEM system including a SEM including an electron gun having an electron output aperture, a sample dish assembly defining a dish aperture for electron communication therethrough, the sample dish assembly including an electron beam permeable, fluid impermeable membrane which at least partially defines a sample enclosure, the sample dish assembly being sealed to the electron gun at the electron output aperture. Preferably, the dish aperture and the electron output aperture are generally aligned in mutual coplanar arrangement. Additionally, the electron gun directs electrons through the electron output aperture in a generally vertically upward direction and the sample dish assembly with the membrane and the dish aperture is facing generally vertically downward.

There is further provided in accordance with yet another preferred embodiment of the present invention a SEM system including a SEM, and a SEM compatible sample container including a sample enclosure including an electron beam permeable, fluid impermeable membrane, and a peripheral enclosure sealed to the membrane and defining with the membrane the sample enclosure, and a sample enclosure closure including quick-connect attachment functionality for sealing engagement with the sample enclosure.

Preferably, the quick-connect attachment functionality includes a threaded connection. Additionally, the peripheral enclosure is at least partially electrically conductive. Alternatively or additionally the SEM system also includes a pressure relief diaphragm associated with the sample enclosure.

In accordance with another preferred embodiment of the present invention the SEM system also includes at least one membrane support grid supporting the electron beam permeable, fluid impermeable, membrane. Preferably, the membrane is formed from a material selected from the group consisting of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, PARLODION, COLLODION, KAPTON, FORMVAR, VINYLEC, BUTVAR, PIOLOFORM, PARYLENE, silicon dioxide, silicon monoxide and carbon. Additionally, the sample enclosure is preassembled and

ready to receive a liquid containing sample therein, following which the sample enclosure closure may be readily sealingly joined thereto by means of the quick-connect attachment functionality.

There is further provided in accordance with yet another preferred embodiment of the present invention a SEM system including a SEM, at least one SEM compatible multiple sample container including an enclosure defining a multiplicity of apertures arranged in an array for electron communication therethrough, and at least one electron beam permeable, fluid impermeable membrane disposed over the multiplicity of apertures, the enclosure and the at least one membrane defining a multiplicity of sample dishes, each including at least a portion of the at least one membrane, and the multiplicity of apertures being arranged with respect to the at least one membrane for electron communication through the at least one membrane with interiors of the sample dishes, and at least one automatic manipulator for automatically positioning the at least one SEM compatible multiple sample container in at least one desired position with respect to the SEM.

Preferably, the SEM system also includes an enclosure cover assembly operative for selective individual sealing of each of the multiplicity of sample dishes.

Additionally, each of the multiplicity of sample dishes is defined by the membrane together with a separate dish wall definer disposed within the enclosure. Alternatively or additionally, the separate dish wall definer is sealed to at least a portion of the at least one membrane.

There is also provided in accordance with yet another preferred embodiment of the present invention a method for performing scanning electron microscopy including placing a sample in a sample enclosure including an electron beam permeable, fluid impermeable membrane, a peripheral enclosure sealed to the membrane and defining with the membrane the sample enclosure, and a sample enclosure closure including quick-connect attachment functionality for sealing engagement with the sample enclosure, sealing the sample enclosure with the sample enclosure closure, placing the sample enclosure in a beam of electrons, and analyzing results of interactions of the beam of electrons with the sample.

Preferably, the method for performing scanning electron microscopy also includes removal of liquid from the sample enclosure prior to the sealing. Additionally,

the method for performing scanning electron microscopy also includes addition of liquid to the sample enclosure prior to the sealing. Alternatively or additionally, the method for performing scanning electron also includes incubation of the sample in the sample enclosure.

In accordance with another preferred embodiment of the present invention the SEM system analysis of the results of interactions of the beam of electrons with the sample is performed by at least one of detection of X-rays, detection of light in the ultraviolet to infrared range, detection of backscattered electrons, and detection of secondary electrons.

There is also provided in accordance with still another preferred embodiment of the present invention a method for performing scanning electron microscopy including placing a sample in a sample enclosure including an electron beam permeable, fluid impermeable membrane, a peripheral enclosure sealed to the membrane and defining with the membrane the sample enclosure, and a sample enclosure closure including quick-connect attachment functionality for sealing engagement with the sample enclosure, positioning a sample positioner arranged to position the sample adjacent to the membrane, sealing the sample enclosure with the sample enclosure closure, placing the sample enclosure in a beam of electrons, and analyzing results of interactions of the beam of electrons with the sample.

In accordance with another preferred embodiment of the present invention the method for performing scanning electron microscopy also includes removal of liquid from the sample enclosure prior to the sealing. Preferably, the method for performing scanning electron microscopy also includes addition of liquid to the sample enclosure prior to the sealing. Additionally, the method for performing scanning electron microscopy also includes incubation of the sample in the sample enclosure.

Alternatively or additionally, analysis of the results of interactions of the beam of electrons with the sample is performed by at least one of detection of X-rays, detection of light in the ultraviolet to infrared range, detection of backscattered electrons, and detection of secondary electrons.

There is further provided in accordance with yet another preferred embodiment of the present invention a tissue sample slicing assembly including a stage, at least one slab placed on the stage and defining a recess for a tissue sample, and a

slicing instrument. Preferably, the at least one slab is operative to provide a predetermined thickness for slicing the tissue sample.

There is further provided in accordance with still another preferred embodiment of the present invention a method for slicing a tissue sample including defining a recess by placing at least one slab on a stage, placing a tissue sample in the recess, and slicing the tissue sample. Preferably, the method also includes selecting the at least one slab to provide a predetermined thickness for the slicing.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be understood and appreciated more fully from the following detailed description, taken in conjunction with the drawings in which: Figs. 1A & 1B are oppositely facing simplified exploded view pictorial illustrations of a disassembled SEM compatible sample container constructed and operative in accordance with a preferred embodiment of the present invention; Figs. 2A & 2B are oppositely facing simplified partially pictorial, partially sectional illustrations of a subassembly of the container of Figs. 1A & 1B Figs. 3A & 3B are oppositely facing simplified exploded view pictorial illustrations of the SEM compatible sample container of Figs. 1A-2B in a partially assembled state; Figs. 4A & 4B are oppositely facing simplified pictorial illustrations of the SEM compatible sample container of Figs. 1A-3B in a fully assembled state; Figs. 5A & 5B are oppositely facing simplified partially pictorial, partially sectional illustrations taken along lines VA-VA and VB-VB, respectively, in Figs. 3A & 3B Figs. 6A, 6B & 6C are three sectional illustrations showing the operative orientation of the SEM compatible sample container of Figs. 1A-SB at three stages of operation; Figs. 7A, 7B, 7C, 7D and 7E are simplified sectional illustrations of cell growth, liquid removal, liquid addition, sealing and insertion into a SEM respectively using the SEM compatible sample container of Figs. 1A-6C ; Figs. 8A, 8B and 8C are simplified sectional illustrations of liquid containing samples, sealing and insertion into a SEM respectively using the SEM compatible sample container of Figs. 1A-6C ; Fig. 9 is a simplified pictorial and sectional illustration of a SEM inspection of a sample using the SEM compatible sample container of Figs. 1A-6C ; Fig. 10 is a greatly enlarged simplified schematic illustration of the SEM inspection of a sample in the context of Fig. 9;

Figs. l lA & l lB are oppositely facing simplified exploded view pictorial illustrations of a disassembled SEM compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention; Figs. 12A & 12B are oppositely facing simplified partially pictorial, partially sectional illustrations of a subassembly of the container of Figs. 11A & 11B ; Figs. 13A & 13B are oppositely facing simplified exploded view pictorial illustrations of the SEM compatible sample container of Figs. 11A-12B in a partially assembled state; Figs. 14A & 14B are oppositely facing simplified pictorial illustrations of the SEM compatible sample container of Figs. 11A-13B in a fully assembled state; Figs. 15A & 15B are oppositely facing simplified partially pictorial, partially sectional illustrations taken along lines XVA-XVA and XVB-XVB, respectively, in Figs. 13A & 13B Figs. 16A, 16B & 16C are three sectional illustrations showing the operative orientation of the SEM compatible sample container of Figs. 11A-15B at three stages of operation; Figs. 17A, 17B, 17C, 17D and 17E are simplified sectional illustrations of cell growth, liquid removal, liquid addition, sealing and insertion into a SEM respectively using the SEM compatible sample container of Figs. 1 lA-16C ; Figs. 18A, 18B and 18C are simplified sectional illustrations of liquid containing samples, sealing and insertion into a SEM respectively using the SEM compatible sample container of Figs. 11A-16C ; Fig. 19 is a simplified pictorial and sectional illustration of a SEM inspection of a sample using the SEM compatible sample container of Figs. 11A-16C ; Fig. 20 is a greatly enlarged simplified schematic illustration of the SEM inspection of a sample in the context of Fig. 19; Figs. 21A and 21B are simplified exploded view illustrations of a pre-microscopy multi-sample holder in use with SEM compatible sample containers of the type shown in Figs. 1A-20 ; Figs. 22A and 22B are simplified illustrations of the pre-microscopy multi-sample holder of Figs. 21A & 21B respectively uncovered and covered in an assembled state;

Figs. 23A, 23B and 23C are simplified illustrations of the pre-microscopy multi-sample holder of Figs. 21A-22B respectively associated with a suction device and pipettes; Figs. 24A, 24B and 24C are simplified illustrations of a microscopy multi-sample holder in use with a SEM compatible sample dish of the type shown in Figs. 1A-10 ; Figs. 25A, 25B and 25C are simplified illustrations of a microscopy multi-sample holder in use with a SEM compatible sample dish of the type shown in Figs. 1 lA-20 ; Figs. 26A and 26B are simplified illustrations of a microscopy multi-sample holder defining a plurality of SEM compatible sample containers in accordance with a preferred embodiment of the present invention; Figs. 27A and 27B are simplified illustrations of a microscopy multi-sample holder defining a plurality of SEM compatible sample containers in accordance with a preferred embodiment of the present invention; Fig. 28 is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with a preferred embodiment of the present invention; Fig. 29 is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with another preferred embodiment of the present invention; Fig. 30 is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with yet another preferred embodiment of the present invention; Figs. 31A&31Bare oppositely facing simplified exploded view pictorial illustrations of a disassembled SEM compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention; Figs. 32A & 32B are oppositely facing simplified partially pictorial, partially sectional illustrations of a subassembly of the container of Figs. 31A & 31B ; Figs. 33A & 33B are oppositely facing simplified exploded view pictorial illustrations of the SEM compatible sample container of Figs. 31A-32B in a partially assembled state;

Figs. 34A & 34B are oppositely facing simplified pictorial illustrations of the SEM compatible sample container of Figs. 31A-33B in a fully assembled state; Figs. 35A & 35B are oppositely facing simplified partially pictorial, partially sectional illustrations taken along lines XXXVA-XXXVA and XXXVB- XXXVB, respectively, in Figs. 33A & 33B Figs. 36A, 36B & 36C are three sectional illustrations showing the operative orientation of the SEM compatible sample container of Figs. 31A-35B at three stages of operation; Fig. 37 is a simplified sectional and pictorial illustration of tissue containing samples and insertion into a SEM using the SEM compatible sample container of Figs. 31A-36C ; Figs. 38A, 38B, 38C and 38D are simplified sectional illustrations showing the operative orientation of a SEM compatible sample container at various stages of operation and insertion into a SEM using the SEM compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention; Fig. 39 is a simplified pictorial and sectional illustration of a SEM inspection of a sample using the SEM compatible sample container of Figs. 31A-37 ; Fig. 40 is a greatly enlarged simplified schematic illustration of the SEM inspection of a sample in the context of Fig. 39; Figs. 41A & 41B are oppositely facing simplified exploded view pictorial illustrations of a disassembled SEM compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention; Figs. 42A & 42B are oppositely facing simplified partially pictorial, partially sectional illustrations of a subassembly of the container of Figs. 41A & 41B Figs. 43A & 43B are oppositely facing simplified exploded view pictorial illustrations of the SEM compatible sample container of Figs. 41A-42B in a partially assembled state; Figs. 44A & 44B are oppositely facing simplified pictorial illustrations of the SEM compatible sample container of Figs. 41A-43B in a fully assembled state;

Figs. 45A & 45B are oppositely facing simplified partially pictorial, partially sectional illustrations taken along lines XLVA-XLVA and XLVB-XLVB, respectively, in Figs. 43A & 43B Figs. 46A, 46B & 46C are three sectional illustrations showing the operative orientation of the SEM compatible sample container of Figs. 41A-45B at three stages of operation; Fig. 47 is a simplified sectional and pictorial illustrations of tissue containing samples and insertion into a SEM using the SEM compatible sample container of Figs. 41A-46C ; Figs. 48A, 48B, 48C and 48D are simplified sectional illustrations showing the operative orientation of a SEM compatible sample container at various stages of operation and insertion into a SEM using the SEM compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention; Fig. 49 is a simplified pictorial and sectional illustration of a SEM inspection of a sample using the SEM compatible sample container of Figs. 41A-46C ; Fig. 50 is a greatly enlarged simplified schematic illustration of the SEM inspection of a sample in the context of Fig. 49; Figs. 51 A, 51B and 51C are simplified illustrations of a microscopy multi-sample holder in use with a SEM compatible sample dish of the type shown in Figs. 31A-40 ; Figs. 52A, 52B and 52C are simplified illustrations of a microscopy multi-sample holder in use with a SEM compatible sample dish of the type shown in Figs. 41A-50; Figs. 53A and 53B are simplified illustrations of a microscopy multi-sample holder defining a plurality of SEM compatible sample containers in accordance with a preferred embodiment of the present invention; Figs. 54A and 54B are simplified illustrations of a microscopy multi-sample holder defining a plurality of SEM compatible sample containers in accordance with a preferred embodiment of the present invention;

Fig. 55 is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with a preferred embodiment of the present invention; Fig. 56 is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with another preferred embodiment of the present invention; Fig. 57 is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with yet another preferred embodiment of the present invention; Figs. 58A & 58B are oppositely facing simplified exploded view pictorial illustrations of a disassembled SEM compatible sample container constructed and operative in accordance with yet another preferred embodiment of the present invention; Figs. 59A & 59B are oppositely facing simplified partially pictorial, partially sectional illustrations of a subassembly of the container of Figs. 58A & 58B Figs. 60A & 60B are oppositely facing simplified exploded view pictorial illustrations of the SEM compatible sample container of Figs. 58A-59B in a partially assembled state; Figs. 61A & 61B are oppositely facing simplified pictorial illustrations of the SEM compatible sample container of Figs. 58A-60B in a fully assembled state ; Figs. 62A & 62B are oppositely facing simplified partially pictorial, partially sectional illustrations taken along lines LXIIA-LXIIA and LXIIB-LXIIB, respectively, in Figs. 60A & 60B Figs. 63A, 63B & 63C are three sectional illustrations showing the operative orientation of the SEM compatible sample container of Figs. 58A-62B at three stages of operation; Figs. 64A, 64B, 64C, 64D and 64E are simplified sectional illustrations of cell growth, liquid removal, liquid addition, sealing and insertion into a SEM respectively using the SEM compatible sample container of Figs. 58A-63C ; Figs. 65A, 65B and 65C are simplified sectional illustrations of liquid containing samples, sealing and insertion into a SEM respectively using the SEM compatible sample container of Figs. 58A-63C ;

Fig. 66 is a simplified pictorial and sectional illustration of a SEM inspection of a sample using the SEM compatible sample container of Figs. 58A-63C; Fig. 67 is a greatly enlarged simplified schematic illustration of the SEM inspection of a sample in the context of Fig. 66; Figs. 68A & 68B are oppositely facing simplified exploded view pictorial illustrations of a disassembled SEM compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention; Figs. 69A & 69B are oppositely facing simplified partially pictorial, partially sectional illustrations of a subassembly of the container of Figs. 68A & 68B Figs. 70A & 70B are oppositely facing simplified exploded view pictorial illustrations of the SEM compatible sample container of Figs. 68A-69B in a partially assembled state; Figs. 71A & 71B are oppositely facing simplified pictorial illustrations of the SEM compatible sample container of Figs. 68A-70B in a fully assembled state; Figs. 72A & 72B are oppositely facing simplified partially pictorial, partially sectional illustrations taken along lines LXXIIA-LXXIIA and LXXIIB- LXXIIB, respectively, in Figs. 70A & 70B Figs. 73A, 73B & 73C are three sectional illustrations showing the operative orientation of the SEM compatible sample container of Figs. 68A-72B at three stages of operation; Figs. 74A, 74B, 74C, 74D and 74E are simplified sectional illustrations of cell growth, liquid removal, liquid addition, sealing and insertion into a SEM respectively using the SEM compatible sample container of Figs. 68A-73C; Figs. 75A, 75B and 75C are simplified sectional illustrations of liquid containing samples, sealing and insertion into a SEM respectively using the SEM compatible sample container of Figs. 68A-73C; Fig. 76 is a simplified pictorial and sectional illustration of a SEM inspection of a sample using the SEM compatible sample container of Figs. 68A-73C ; Fig. 77 is a greatly enlarged simplified schematic illustration of the SEM inspection of a sample in the context of Fig. 76;

Figs. 78A, 78B and 78C are simplified illustrations of a microscopy multi-sample holder in use with a SEM compatible sample dish of the type shown in Figs. 58A-67; Figs. 79A, 79B and 79C are simplified illustrations of a microscopy multi-sample holder in use with a SEM compatible sample dish of the type shown in Figs. 68A-77; Figs. 80A and 80B are simplified illustrations of a microscopy multi-sample holder defining a plurality of SEM compatible sample containers in accordance with a preferred embodiment of the present invention; Figs. 81A and 81B are simplified illustrations of a microscopy multi-sample holder defining a plurality of SEM compatible sample containers in accordance with a preferred embodiment of the present invention; Fig. 82 is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with a preferred embodiment of the present invention; Fig. 83 is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with another preferred embodiment of the present invention; Fig. 84 is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with yet another preferred embodiment of the present invention; Fig. 85 is a simplified partially pictorial and partially sectional illustration of SEM inspection of a sample constructed and operative in accordance with another preferred embodiment of the present invention; Figs. 86A and 86B are simplified partially pictorial and partially sectional illustration of a tissue sample slicing assembly constructed and operative in accordance with a preferred embodiment of the present invention; and Fig. 87A and 87B are simplified partially pictorial and partially sectional illustration of a tissue sample slicing assembly constructed and operative in accordance with a preferred embodiment of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Reference is now made to Figs. 1A-5B, which are oppositely facing simplified exploded view pictorial illustrations of a disassembled scanning electron microscope (SEM) compatible sample container constructed and operative in accordance with a preferred embodiment of the present invention. As seen in Figs. 1A & 1B, the SEM compatible sample container comprises first and second mutually threaded enclosure elements, respectively designated by reference numerals 100 and 102, arranged for enhanced ease and speed of closure. Enclosure elements 100 and 102 are preferably molded of plastic and coated with a conductive metal coating.

First enclosure element 100 preferably defines a liquid sample enclosure and has a base surface 104 having a generally central aperture 106. An electron beam permeable, fluid impermeable, membrane subassembly 108, shown in detail in Figs. 2A and 2B, is seated inside enclosure element 100 against and over aperture 106, as shown in Figs. 3A & 3B and 5A & 5B. A sample dish comprising subassembly 108 suitably positioned in enclosure element 100 is designated by reference numeral 109, as shown in Figs. 3A-5B.

Turning additionally to Figs. 2A and 2B, it is seen that an electron beam permeable, fluid impermeable, membrane 110, preferably a polyimide membrane, such as Catalog No. LWN00033, commercially available from Moxtek Inc. of Orem, UT, U. S. A. , is adhered, as by an adhesive, to a mechanically supporting grid 112. Grid 112, which is not shown to scale, is preferably Catalog No. BM 0090-01, commercially available from Buckbee-Mears of Cortland, N. Y. , U. S. A. , and the adhesive is preferably Catalog No. NOA61, commercially available from Norland Products Inc. of Cranbury, NJ, U. S. A. A liquid sample enclosure defining ring 114 is adhered to electron beam permeable, fluid impermeable, membrane 110, preferably by an adhesive, such as Catalog No. NOA61, commercially available from Norland Products Inc. of Cranbury, NJ, U. S. A. Ring 114 is preferably formed of PMMA (polymethyl methacrylate), such as Catalog No. 692106001000, commercially available from Irpen of Barcelona, Spain, and preferably defines a liquid sample enclosure with a volume of approximately 20 microliters and a height of approximately 2 mm. Preferably ring 114 is configured to define a liquid sample enclosure 116 having inclined walls.

Alternatively, membrane 110 may be formed from polyamide, polyamide-imide, polyethylene, polypynole, PARLODION, COLLODION, KAPTON, FORMVAR, VINYLEC, BUTVAR, PIOLOFORM, PARYLENE, silicon dioxide, silicon monoxide or carbon, or any combination thereof or any other suitable material.

An O-ring 118 is preferably disposed between ring 114 and an interior surface 120 of second enclosure element 102. O-ring 118 is operative, when enclosure elements 100 and 102 are in tight threaded engagement, to obviate the need for the threaded engagement of elements 100 and 102 to be a sealed engagement.

Second enclosure element 102 preferably is formed with a generally central stub 122, which is arranged to be seated in a suitable recess (not shown) in a specimen stage of a scanning electron microscope. It is a particular feature of the present invention that the container, shown in Figs. 1A-10, is sized and operative with conventional stub recesses in conventional scanning electron microscopes and does not require any modification thereof whatsoever. It is appreciated that various configurations and sizes of stubs may be provided so as to fit various scanning electron microscopes.

Enclosure elements 100 and 102 are preferably also provided with respective radially extending positioning and retaining protrusions 124 and 125, to enable the container to be readily seated in a suitable multi-container holder and also to assist users in threadably opening and closing the enclosure elements 100 and 102.

Preferably, the mutual azimuthal positioning of the protrusions 124 and 125 on respective enclosure elements 100 and 102 is such that mutual azimuthal alignment therebetween indicates a desired degree of threaded closure therebetween, as shown in Figs. 4A and 4B.

It is appreciated that in another embodiment of the present invention the sample dish may include enclosures 100 and 102.

Reference is now made to Figs. 6A, 6B & 6C, which are three sectional illustrations showing the operative orientation of the SEM compatible sample container of Figs. 1A-5B at three stages of operation. Fig. 6A shows the container of Figs. 1A- 5B containing a liquid sample 130 and arranged in the orientation shown in Fig. 1B, prior to threaded closure enclosure elements 100 and 102. It is noted that the liquid sample does not flow out of the liquid sample enclosure 116 due to surface tension. The

electron beam permeable, fluid impermeable, membrane 110 is seen in Fig. 6A to be generally planar.

Fig. 6B shows the container of Fig. 6A immediately following full threaded engagement between enclosure elements 100 and 102, producing sealing of the liquid sample enclosure 116 from the ambient. It is seen that the electron beam permeable, fluid impermeable, membrane 110 and its supporting grid 112 bow outwardly due to pressure buildup in the liquid sample enclosure 116 as the result of sealing thereof in this manner.

Fig. 6C illustrates the container of Fig. 6B, when placed in an evacuated environment of a SEM, typically at a vacuum of 10-2-10'6 millibars. It is seen that in this environment, the electron beam permeable, fluid impermeable, membrane 110 and support grid 112 bow outwardly to a greater extent than in the ambient environment of Fig. 6B and further that the electron beam permeable, fluid impermeable, membrane 110 tends to be forced into and through the interstices of grid 112 to a greater extent than occurs in the ambient environment of Fig. 6B.

Reference is now made to Figs. 7A, 7B, 7C, 7D and 7E, which are simplified sectional illustrations of cell growth, liquid removal, liquid addition, sealing and insertion into a SEM respectively using the SEM compatible sample container of Figs. 1A-6C. Turning to Fig. 7A, which illustrates a typical cell culture situation, it is seen that the enclosure element 100 having disposed therewithin subassembly 108 is in the orientation shown in Fig. 1A and cells 140 in a liquid medium 142 are located within liquid sample enclosure 116, the cells 140 lying against the electron beam permeable, fluid impermeable, membrane 110.

Fig. 7B shows removal of liquid from liquid sample enclosure 116, typically by aspiration, and Fig. 7C shows addition of liquid to liquid sample enclosure 116. It is appreciated that multiple occurrences of liquid removal and addition may take place with respect to a sample within liquid sample enclosure 116. Preferably, the apparatus employed for liquid removal and addition is designed or equipped such as to prevent inadvertent rupture of the electron beam permeable, fluid impermeable, membrane 110.

Fig. 7D illustrates closing of the container containing the cells 140, seen in Fig. 7C, in a liquid medium 142. Fig. 7E shows the closed container, in the

orientation of Fig. 1B being inserted onto a stage 144 of a SEM 146. It is appreciated that there exist SEMs wherein the orientation of the container is opposite to that shown in Fig. 7E.

Figs. 7A-7D exemplify a situation wherein at least a portion of a liquid containing sample remains in contact with the electron beam permeable, fluid impermeable, membrane 110 notwithstanding the addition or removal of liquid from liquid sample enclosure 116. This situation may include situations wherein part of the sample is adsorbed or otherwise adhered to the electron beam permeable, fluid impermeable, membrane 110. Examples of liquid containing samples may include cell cultures, blood, bacteria and acellular material.

Reference is now made to Figs. 8A, 8B and 8C which are simplified sectional illustrations of liquid containing samples in contact with the electron beam permeable, fluid impermeable, membrane 110, sealing and insertion into a SEM respectively using the SEM compatible sample container of Figs. 1A-6C. Figs. 8A- 8C exemplify a situation wherein at least a portion of a liquid containing sample 160 is in contact with the electron beam permeable, fluid impermeable, membrane 110 but is not adhered thereto. Examples of liquid containing samples may include various emulsions and suspensions such as milk, cosmetic creams, paints, inks, and pharmaceuticals in liquid form. It is seen that the enclosure element 100 in Figs. 8A-8B, having disposed therewithin subassembly 108, is in the orientation shown in Fig. 1A.

Fig. 8B illustrates closing of the container containing the sample 160.

Fig. 8C shows the closed container, in the orientation of Fig. 1B, being inserted onto a stage 144 of a SEM 146. It is appreciated that there exist SEMs wherein the orientation of the container is opposite to that shown in Fig. 8C.

Reference is now made to Fig. 9, which is a simplified pictorial and sectional illustration of SEM inspection of a sample using the SEM compatible sample container of Figs. 1A-6C. As seen in Fig. 9, the container, here designated by reference numeral 170, is shown positioned on stage 144 of a SEM 146 such that an electron beam 172, generated by the SEM, passes through electron beam permeable, fluid impermeable, membrane 110 and impinges on a liquid containing sample 174 within container 170. Backscattered electrons from sample 174 pass through electron beam permeable, fluid impermeable, membrane 110 and are detected by a detector 176,

forming part of the SEM. One or more additional detectors, such as a secondary electron detector 178 may also be provided. An X-ray detector (not shown) may also be provided for detecting X-ray radiation emitted by the sample 174 due to electron beam excitation thereof.

Reference is now made additionally to Fig. 10, which schematically illustrates some details of the electron beam interaction with the sample 174 in container 170 in accordance with a preferred embodiment of the present invention. It is noted that the present invention enables high contrast imaging of features which are distinguished from each other by their average atomic number, as illustrated in Fig. 10. In Fig. 10 it is seen that nucleoli 180, having a relatively high average atomic number, backscatter electrons more than the surrounding nucleoplasm 182.

It is also noted that in accordance with a preferred embodiment of the present invention, imaging of the interior of the sample to a depth of up to approximately 2 microns is achievable for electrons having an energy level of less than 50KeV, as seen in Fig. 10, wherein nucleoli 180 disposed below electron beam permeable, fluid impermeable, membrane 110 are imaged.

Reference is now made to Figs. 1lA-158, which are oppositely facing simplified exploded view pictorial illustrations of a disassembled scanning electron microscope (SEM) compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention. As seen in Figs. 11A & 11B, the SEM compatible sample container comprises first and second mutually threaded enclosure elements, respectively designated by reference numerals 200 and 202, arranged for enhanced ease and speed of closure. Enclosure elements 200 and 202 are preferably molded of plastic and coated with a conductive metal coating.

First enclosure element 200 preferably defines a liquid sample enclosure and has a base surface 204 having a generally central aperture 206. An electron beam permeable, fluid impermeable, membrane subassembly 208, shown in detail in Figs.

12A and 12B, is seated inside enclosure element 200 against and over aperture 206, as shown in Figs. 13A & 13B and 15A & 15B. A sample dish comprising subassembly 208 suitably positioned in enclosure element 200 is designated by reference numeral 209, as shown in Figs. 13A-15B.

Turning additionally to Figs. 12A and 12B, it is seen that an electron beam permeable, fluid impermeable, membrane 210, preferably a polyimide membrane, such as Catalog No. LWN00033, commercially available from Moxtek Inc. of Orem, <BR> <BR> UT, U. S. A. , is adhered, as by an adhesive, to a mechanically supporting grid 212. Grid 212, which is not shown to scale, is preferably Catalog No. BM 0090-01, commercially <BR> <BR> available from Buckbee-Mears of Cortland, N. Y. , U. S. A. , and the adhesive is preferably Catalog No. NOA61, commercially available from Norland Products Inc. of Cranbury, NJ, U. S. A. A liquid sample enclosure defining ring 214 is adhered to electron beam permeable, fluid impermeable, membrane 210, preferably by an adhesive, such as Catalog No NOA61, commercially available from Norland Products Inc. of Cranbury, NJ, U. S. A.. Ring 214 is preferably formed of PMMA (polymethyl methacrylate), such as Catalog No. 692106001000, commercially available from Irpen of Barcelona, Spain, and preferably defines a liquid sample enclosure with a volume of approximately 20 microliters and a height of approximately 2 mm. Preferably ring 214 is configured to define a liquid sample enclosure 216 having inclined walls.

A diaphragm 218 is preferably disposed between ring 214 and an interior surface 219 of second enclosure element 202. Diaphragm 218 is preferably integrally formed of an O-ring portion 220 to which is sealed an expandable sheet portion 221.

The diaphragm 218 is preferably molded of silicon rubber having a Shore hardness of about 50 and the sheet portion 221 preferably has a thickness of 0.2-0. 3 mm.

Diaphragm 218 is operative, when enclosure elements 200 and 202 are in tight threaded engagement, to obviate the need for the threaded engagement of elements 200 and 202 to be a sealed engagement and to provide dynamic and static pressure relief.

Second enclosure element 202 preferably is formed with a generally central stub 222, having a throughgoing bore 223, which stub is arranged to be seated in a suitable recess (not shown) in a specimen stage of a scanning electron microscope.

Bore 223 enables diaphragm 218 to provide pressure relief by defining a fluid communication channel between one side of the diaphragm 218 and the environment in which the (SEM) compatible sample container is located. It is a particular feature of the present invention that the container, shown in Figs. 1lA-20, is sized and operative with conventional stub recesses in conventional scanning electron microscopes and does not require any modification thereof whatsoever. It is appreciated that various

configurations and sizes of stubs may be provided so as to fit various scanning electron microscopes.

Enclosure elements 200 and 202 are preferably also provided with respective radially extending positioning and retaining protrusions 224 and 225, to enable the container to be readily seated in a suitable multi-container holder and also to assist users in threadably opening and closing the enclosure elements 200 and 202.

Preferably, the mutual azimuthal positioning of the protrusions 224 and 225 on respective enclosure elements 200 and 202 is such that mutual azimuthal alignment therebetween indicates a desired degree of threaded closure therebetween, as shown in Figs. 14A and 14B.

Reference is now made to Figs. 16A, 16B & 16C, which are three sectional illustrations showing the operative orientation of the SEM compatible sample container of Figs. 1 lA-15B at three stages of operation. Fig. 16A shows the container of Figs. 11A-15B containing a liquid sample 230 and arranged in the orientation shown in Fig. 11B, prior to threaded closure enclosure elements 200 and 202. It is noted that the liquid sample does not flow out of the liquid sample enclosure 216 due to surface tension. The electron beam permeable, fluid impermeable, membrane 210 is seen in Fig. 16A to be generally planar.

Fig. 16B shows the container of Fig. 16A immediately following full threaded engagement between enclosure elements 200 and 202, producing sealing of the liquid sample enclosure 216 from the ambient. It is seen that the diaphragm 218 bows outwardly due to pressure buildup in the liquid sample enclosure 216 as the result of sealing thereof in this manner. In this embodiment, electron beam permeable, fluid impermeable, membrane 210 and its supporting grid 212 also bow outwardly due to pressure buildup in the liquid sample enclosure 216 as the result of sealing thereof in this manner, however to a significantly lesser extent, due to the action of diaphragm 218. This can be seen by comparing Fig. 16B with Fig. 6B.

Fig. 16C illustrates the container of Fig. 16B, when placed in an evacuated environment of a SEM, typically at a vacuum of 10-2 10-6 millibars. It is seen that in this environment, the diaphragm 218 bows outwardly to a greater extent than in the ambient environment of Fig. 16B and that electron beam permeable, fluid impermeable, membrane 210 and support grid 212 also bow outwardly to a greater

extent than in the ambient environment of Fig. 16B, but to a significantly lesser extent than in the embodiment of Fig. 6C, due to the action of diaphragm 218. This can be seen by comparing Fig. 16C with Fig. 6C.

It is also noted that the electron beam permeable, fluid impermeable, membrane 210 tends to be forced into and through the interstices of grid 212 to a greater extent than occurs in the ambient environment of Fig. 16B but to a significantly lesser extent than in the embodiment of Fig. 6C, due to the action of diaphragm 218. This can also be seen by comparing Fig. 16C with Fig. 6C.

Reference is now made to Figs. 17A, 17B, 17C, 17D and 17E, which are simplified sectional illustrations of cell growth, liquid removal, liquid addition, sealing and insertion into a SEM respectively using the SEM compatible sample container of Figs. 11A-16C. Turning to Fig. 17A, which is identical to Fig. 7A and illustrates a typical cell culture situation, it is seen that the enclosure element 200 having disposed therewithin subassembly 208 is in the orientation shown in Fig. 11A and cells 240 in a liquid medium 242 are located within liquid sample enclosure 216, the cells 240 lying against the electron beam permeable, fluid impermeable, membrane 210.

Fig. 17B, which is identical to Fig. 7B, shows removal of liquid from liquid sample enclosure 216, typically by aspiration, and Fig. 17C, which is identical to Fig. 7C, shows addition of liquid to liquid sample enclosure 216. It is appreciated that multiple occurrences of liquid removal and addition may take place with respect to a sample within liquid sample enclosure 216. Preferably, the apparatus employed for liquid removal and addition is designed or equipped such as to prevent inadvertent rupture of the electron beam permeable, fluid impermeable, membrane 210.

Fig. 17D illustrates closing of the container containing the cells 240, seen in Fig. 17C, in a liquid medium 242. Fig. 17E shows the closed container, in the orientation of Fig. 11B being inserted onto a stage 244 of a SEM 246. It is appreciated that there exist SEMs wherein the orientation of the container is opposite to that shown in Fig. 17E.

Figs. 17A-17D exemplify a situation wherein at least a portion of a liquid containing sample remains in contact with the electron beam permeable, fluid impermeable, membrane 210 notwithstanding the addition or removal of liquid from liquid sample enclosure 216. This situation may include situations wherein part of the

sample is adsorbed or otherwise adhered to the electron beam permeable, fluid impermeable, membrane 210. Examples of liquid containing samples may include cell cultures, blood, bacteria and acellular material.

Reference is now made to Figs. 18A, 18B and 18C which are simplified sectional illustrations of liquid containing samples in contact with the electron beam permeable, fluid impermeable, membrane 210, sealing and insertion into a SEM respectively using the SEM compatible sample container of Figs. 11A-16C. Figs. 18A - 18C exemplify a situation wherein at least a portion of a liquid containing sample 260 is in contact with the electron beam permeable, fluid impermeable, membrane 210 but is not adhered thereto. Examples of liquid containing samples may include various emulsions and suspensions such as milk, cosmetic creams, paints, inks, and pharmaceuticals in liquid form. It is seen that the enclosure element 200, having disposed therewithin subassembly 208 in Figs. 8A-8B, is in the orientation shown in Fig. 11A. Fig. 18A is identical to Fig. 8A.

Fig. 18B illustrates closing of the container containing the sample 260.

Fig. 18C shows the closed container, in the orientation of Fig. 11B, being inserted onto a stage 244 of a SEM 246. It is appreciated that there exist SEMs wherein the orientation of the container is opposite to that shown in Fig. 18C.

Reference is now made to Fig. 19, which is a simplified pictorial and sectional illustration of SEM inspection of a sample using the SEM compatible sample container of Figs. 11A-16C. As seen in Fig. 19, the container, here designated by reference numeral 270, is shown positioned on stage 244 of a SEM 246 such that an electron beam 272, generated by the SEM, passes through electron beam permeable, fluid impermeable, membrane 210 and impinges on a liquid containing sample 274 within container 270. Backscattered electrons from sample 274 pass through electron beam permeable, fluid impermeable, membrane 210 and are detected by a detector 276, forming part of the SEM. One or more additional detectors, such as a secondary electron detector 278 may also be provided. An X-ray detector (not shown) may also be provided for detecting X-ray radiation emitted by the sample 274 due to electron beam excitation thereof.

Reference is now made additionally to Fig. 20, which schematically illustrates some details of the electron beam interaction with the sample 274 in container

270 in accordance with a preferred embodiment of the present invention. It is noted that the present invention enables high contrast imaging of features which are distinguished from each other by their average atomic number, as illustrated in Fig. 20. In Fig. 20 it is seen that nucleoli 280 having a relatively high average atomic number, backscatter electrons more than the surrounding nucleoplasm 282.

It is also noted that in accordance with a preferred embodiment of the present invention, imaging of the interior of the sample to a depth of up to approximately 2 microns is achievable for electrons having an energy level of less than 50KeV, as seen in Fig. 20, wherein nucleoli 280 disposed below electron beam permeable, fluid impermeable, membrane 210 are imaged.

Reference is now made to Figs. 21A and 21B, which are simplified exploded view illustrations of a pre-microscopy multi-sample holder in use with SEM compatible sample containers of the type shown in Figs. 1A-20 and to Figs. 22A and 22B which are simplified illustrations of the pre-microscopy multi-sample holder of Figs. 21A & 21 B respectively uncovered and covered in an assembled state.

As seen in Figs. 21A and 21B, the pre-microscopy multi-sample holder preferably comprises a base 300, a top element 302 and a cover 304. Cover 304 is preferably provided to maintain sterility within the interior of the pre-microscopy multi-sample holder.

The base 300 is preferably injection molded of a plastic material and defines an array of container support locations 306. Each container support location 306 is preferably defined by a recess 308 having a light transparent bottom wall through which light microscopy may take place. Adjacent to each recess 308 there is preferably formed a pair of mutually aligned pairs of upstanding mutually spaced protrusions 310 arranged to receive protrusions, designated by reference numeral 124 in Figs. 1A-4B, on enclosure elements, designated by reference numeral 100 in Figs. 1A-8C, thereby fixing the azimuthal alignment thereof.

Base 300 preferably also defines a plurality of liquid reservoirs 312 which are adapted to hold liquid used to maintain a desired level of humidity in the interior of the pre-microscopy multi-sample holder. Base 300 is preferably formed with a floor 320. Top element 302 is arranged for removable snap-fit engagement with base 300 so as to retain sample dishes, designated by reference numeral 109 in Figs.

3A-5B, in a desired array on base 300. Top element 302 is formed with a planar surface 322 having an array of apertures 324 which are arranged to overlie the sample dishes 109 when seated at container support locations 306. The size of apertures 324 is preferably selected to be less than the size of the enclosure elements 100, so as to prevent the sample dishes 109 from passing therethrough. Planar surface 322 preferably also includes apertures 326 communicating with liquid reservoirs 312.

Top element 302 also provides positioning guides 328 and dummy apertures 330 for use by a suction device in conjunction therewith, as described hereinbelow with reference to Fig. 23A and 23B. Combination dummy apertures 332 are also provided. Only part of each aperture 332 covers a liquid reservoir 312, while the reminder of each aperture 332 serves as a dummy aperture for a suction device.

Cover 304 is provided to maintain sterility of the interior of the pre-microscopy multi-sample holder. Cover 304 is preferably transparent to light, as illustrated in Fig. 22B. The pre-microscopy multi-sample holder of Figs. 21A-22B is preferably dimensioned so as to be compatible with conventional cell biology equipment, such as light microscopes, centrifuges and automated positioning devices.

Preferred dimensions are 85 mm x 127 mm.

Reference is now made to Figs. 23A, 23B and 23C, which are simplified illustrations of the pre-microscopy multi-sample holder of Figs. 21A-22B respectively associated with a suction device and pipettes. Turning to Fig. 23A, it is seen that the suction device, here designated by reference numeral 350, comprises a manifold 352 coupled via a conduit 354 to a source of suction. The manifold 352 preferably communicates with a linear array of uniformly spaced needles 356. A pair of spacers 358 is attached to the manifold 352 or is integrally formed therewith. Spacers 358 are arranged in line with the linear array of needles 356. These spacers 358 preferably engage floor 320 of base 300 at intermediate adjacent positioning guides 328 on opposite sides of the top element 302. The spacers 358 ensure that the needles 356 do not engage electron beam permeable, fluid impermeable, membrane, designated by reference numeral 110 in Figs. lA-10.

As seen in Fig. 23A, the container support locations 306 are arranged in staggered rows on the pre-microscopy multi-sample holder. Thus, as seen in Fig. 23B, in every row, three of the needles 356 engage apertures 324, two of the needles 356

engage dummy apertures 330 and one of the needles 356 engages the part of an aperture 332 which serves as a dummy aperture.

Fig. 23C illustrates addition of liquid to individual sample dishes 109 by means of conventional pipettes 360. Collar elements 362 may be provided for use in association with pipettes 360 to prevent inadvertent engagement of the pipettes with electron beam permeable, fluid impermeable, membrane, designated by reference numeral 110 in Figs. 1 A-10.

Reference is now made to Figs. 24A, 24B and 24C, which are simplified illustrations of a microscopy multi-sample holder in use with a SEM compatible sample dish of the type shown in Figs. 1A-10. As seen in Fig. 24A, the microscopy multi-sample holder preferably comprises a base 400 and a sealing cover 404. The base 400 is preferably injection molded of a plastic material and defines an array of dish support locations 406. Each dish support location 406 is preferably defined by an aperture 408 through which SEM microscopy may take place. Adjacent to each aperture 408 there is preferably formed a pair of mutually aligned pairs of upstanding mutually spaced protrusions 410 arranged to receive protrusions 424 on sample dishes 425.

Sample dishes 425 may be generally identical to sample dishes 109, shown in Figs. 3A -5B, but do not require any threading or other attachment mechanism.

Base 400 may define a plurality of liquid reservoirs 412 which are adapted to hold liquid used to maintain a desired level of humidity in the interior of the microscopy multi-sample holder.

Sealing cover 404 is arranged for individual sealing engagement with each of sample dishes 425. Preferably sealing cover 404 is provided on the underside thereof with an array of 0-rings 426, shown in Fig 24C, sealed thereto and arranged so as to sealingly engage a top rim surface of each of sample dishes 425, when the sealing cover 404 is in place, preferably in removable snap-fit engagement with base 400.

Fig. 24B shows the apparatus of Fig. 24A with one sample dish 425 positioned at a dish support location 406 in base 400. Fig. 24C shows sealing cover 404 in snap fit engagement with base 400, thereby providing individual sealing of each of sample dishes 425 by means of O-ring 426 and a portion of sealing cover 404 circumscribed thereby.

Reference is now made to Figs. 25A, 25B and 25C, which are simplified illustrations of a microscopy multi-sample holder in use with a SEM compatible sample dish of the type shown in Figs. 11A-20. As seen in Fig. 25A, the microscopy multi-sample holder preferably comprises a base 450 and a sealing cover 454. The base 450 is preferably injection molded of a plastic material and defines an array of dish support locations 456. Each dish support location 456 is preferably defined by an aperture 458 through which SEM microscopy may take place. Adjacent to each aperture 458 there is preferably formed a pair of mutually aligned pairs of upstanding mutually spaced protrusions 460 arranged to receive protrusions 474 on sample dishes 475.

Sample dishes 475 may be generally identical to sample dishes 209, shown in Figs. 13A - 15B, but do not require any threading or other attachment mechanism.

Base 450 may define a plurality of liquid reservoirs 462 which are adapted to hold liquid used to maintain a desired level of humidity in the interior of the microscopy multi-sample holder.

Sealing cover 454 is arranged for individual sealing engagement of each of sample dishes 475 with a diaphragm 476, shown in Fig. 25C, which is sealingly mounted over an aperture 478 formed in sealing cover 454. Preferably an array of diaphragms 476, which may be identical to diaphragms 218 described hereinabove with reference to Figs. 11A-20, is provided on the underside of sealing cover 454. The individual diaphragms 476 are arranged so as to sealingly engage a top rim surface of each of sample dishes 475, when the sealing cover 454 is in place, preferably in removable snap-fit engagement with base 450.

Fig. 25B shows the apparatus of Fig. 25A with one sample dish 475 positioned at a dish support location 456 in base 450. Fig. 25C shows sealing cover 454 in snap fit engagement with base 450, thereby providing individual sealing of each of sample dishes 475 by means of diaphragm 476.

Reference is now made to Figs. 26A and 26B, which are simplified illustrations of a microscopy multi-sample holder defining a plurality of SEM compatible sample containers in accordance with a preferred embodiment of the present invention. As seen in Fig. 26A, the microscopy multi-sample holder preferably comprises a base 500 and a sealing cover 504. The base 500 is preferably injection molded of a plastic material and defines an array of sample containers 506. Each sample

container 506 preferably includes an aperture 508 through which SEM microscopy may take place. An electron beam permeable, fluid impermeable, membrane 510, shown in Fig. 26B, is sealed over each aperture 508. Membrane 510 is preferably identical to membrane 110 described hereinabove with reference to Figs. 1A-10. Sealing cover 504 preferably is arranged for individual sealing engagement with each of sample containers 506.

Fig. 26B shows the apparatus of Fig. 26A in sealed engagement, thereby providing individual sealing of each of sample containers 506.

Reference is now made to Figs. 27A and 27B, which are simplified illustrations of a microscopy multi-sample holder defining a plurality of SEM compatible sample containers in accordance with a preferred embodiment of the present invention. As seen in Fig. 27A, the microscopy multi-sample holder preferably comprises a base 550 and a sealing cover 554. The base 550 is preferably injection molded of a plastic material and defines an array of sample containers 556. Each sample container 556 preferably includes an aperture 558 through which SEM microscopy may take place. An electron beam permeable, fluid impermeable, membrane 560, shown in Fig. 27B, is sealed over each aperture 558. Membrane 560 is preferably identical to membrane 210 described hereinabove with reference to Figs. 11A-20. Sealing cover 554, preferably a diaphragm formed of resilient sheet material such as silicon rubber of 0. 2-0.3 mm in thickness and having a Shore hardness of about 50, is arranged for individual sealing engagement with each of sample containers 556.

Fig. 27B shows the apparatus of Fig. 27A in sealed engagement, thereby providing individual sealing of each of sample containers 556.

Reference is now made to Fig. 28, which is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with a preferred embodiment of the present invention. As seen in Fig. 28, a plurality of pre-microscopy multi-sample holders 600, each containing a multiplicity of SEM compatible sample containers 602 of the type shown in Figs. 1A-20, is shown in an incubator 604. Preferably, light microscopy inspection of the samples in containers 602 is carried out while the containers 602 are mounted in holder 600, as indicated at reference numeral 606, in order to identify samples of interest. Preferably an inverted light microscope 608 is employed for this purpose.

Preferably automated positioning systems, such as robotic arms, as shown, are used for conveying the pre-microscopy multi-sample holders 600 and the containers 602 throughout the system, it being appreciated that manual intervention may be employed at one or more stages as appropriate.

Thereafter, individual containers 602 are removed from holders 600 and placed on a removable electron microscope specimen stage 610, which is subsequently introduced into a scanning electron microscope 612. The resulting image may be inspected visually by an operator and/or analyzed by conventional image analysis functionality, typically embodied in a computer 614.

Reference is now made to Fig. 29, which is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with another preferred embodiment of the present invention. As seen in Fig. 29, a plurality of microscopy multi-sample holders 650, each containing a multiplicity of SEM compatible sample dishes 652 of either of the types shown in Figs. 24A-25C, is shown in an incubator 654. Preferably, light microscopy inspection of the samples in sample dishes 652 is carried out while the sample dishes are mounted in holder 650, as indicated at reference numeral 656, in order to identify samples of interest. Preferably an inverted light microscope 658 is employed for this purpose.

Preferably automated positioning systems, such as robotic arms, as shown, are used for conveying the microscopy multi-sample holders 650 containing sample dishes 652 throughout the system, it being appreciated that manual intervention may be employed at one or more stages as appropriate.

Thereafter, holders 650 are placed on an electron microscope specimen stage 660, which is subsequently introduced into a scanning electron microscope 662.

The resulting images may be inspected visually by an operator and/or analyzed by conventional image analysis functionality, typically embodied in a computer 664.

Reference is now made to Fig. 30, which is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with yet another preferred embodiment of the present invention. As seen in Fig. 30, a plurality of microscopy multi-sample holders 670, each defining a multiplicity of SEM compatible sample containers 672, as shown in any of Figs. 26A-27B, is seen in an incubator 674.

Preferably, light microscopy inspection of the samples in sample containers 672 is

carried out holder-wise, as indicated at reference numeral 676, preferably in order to identify samples of interest. Preferably an inverted light microscope 678 is employed for this purpose.

Preferably automated positioning systems, such as robotic arms, as shown, are used for conveying the microscopy multi-sample holders 670 throughout the system, it being appreciated that manual intervention may be employed at one or more stages as appropriate.

Thereafter, holders 670 are placed on an electron microscope specimen stage 680, which is subsequently introduced into a scanning electron microscope 682.

The resulting images may be inspected visually by an operator and/or analyzed by conventional image analysis functionality, typically embodied in a computer 684.

Reference is now made to Figs. 31A-35B, which are oppositely facing simplified exploded view pictorial illustrations of a disassembled scanning electron microscope (SEM) compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention. As seen in Figs. 31A & 31B, the SEM compatible sample container comprises first and second threaded enclosure elements, respectively designated by reference numerals 1100 and 1102, arranged for enhanced ease and speed of closure. Enclosure elements 1100 and 1102 are preferably molded of plastic and coated with a conductive metal coating.

First enclosure element 1100 preferably defines a sample enclosure and has a base surface 1104 having a generally central aperture 1106. An electron beam permeable, fluid impermeable, membrane subassembly 1108, shown in detail in Figs.

32A and 32B, is seated inside enclosure element 1100 against and over aperture 1106, as shown in Figs. 33A & 33B and 35A & 35B. A sample dish comprising subassembly 1108 suitably positioned in enclosure element 1100 is designated by reference numeral 1109, as shown in Figs. 33A-35B.

Turning additionally to Figs. 32A and 32B, it is seen that an electron beam permeable, fluid impermeable, membrane 1110, preferably a polyimide membrane, such as Catalog No. LWN00033, commercially available from Moxtek Inc. <BR> <BR> of Orem, UT, U. S. A. , is adhered, as by an adhesive, to a mechanically supporting grid 1112. Grid 1112, which is not shown to scale, is preferably Catalog No. BM 0090-01, <BR> <BR> commercially available from Buckbee-Mears of Cortland, N. Y. , U. S. A. , and the

adhesive is preferably Catalog No. NOA61, commercially available from Norland Products Inc. of Cranbury, NJ, U. S. A. A sample enclosure defining ring 1114 is adhered to electron beam permeable, fluid impermeable, membrane 1110, preferably by an adhesive, such as Catalog No. NOA61, commercially available from Norland Products Inc. of Cranbury, NJ, U. S. A. Ring 1114 is preferably formed of PMMA (polymethyl methacrylate), such as Catalog No. 692106001000, commercially available from Irpen of Barcelona, Spain, and preferably defines a sample enclosure with a volume of approximately 20 microliters and a height of approximately 2 mm. Preferably ring 1114 is configured to define a sample enclosure 1116 having inclined walls.

A first O-ring 1118 is preferably disposed between an interior surface 1120 of second enclosure element 1102 and a connecting element 1122. Connecting element 1122 is preferably molded of plastic and coated with a conductive metal coating. A second O-ring 1123 is preferably disposed between connecting element 1122 and ring 1114 of subassembly 1108. 0-rings 1118 and 1123 are operative, when enclosure elements 1100 and 1102 and connecting element 1122 are in tight threaded engagement, to obviate the need for the threaded engagement of elements 1100 and 1102 and connecting element 1122 to be a sealed engagement.

Connecting element 1122 preferably has a recess 1124. Connecting element 1122 is also formed with a protrusion 1126, seen in Figs. 35A & 35B, protruding into recess 1124.

A positioner 1128 is preferably comprised of two upright flexible projections 1130, each with a ridge 1132 formed on an end 1134 of the projections 1130. Positioner 1128 is preferably molded of plastic. Projections 1130 press against each other when inserted into recess 1124 of connecting element 1122 and then snap back to an upright position once ridges 1132 are seated on the protrusion 1126 of connecting element 1122, as shown in Figs. 35A & 35B.

Positioner 1128 is preferably also provided with respective radially extending positioning and retaining protrusions 1136 extending from a rim 1137.

Positioning and retaining protrusions 1136 are seated in apertures 1140 formed in the inclined walls of sample enclosure 1116 of ring 1114 to prevent rotation of positioner 1128.

A coil spring 1142 is disposed on positioner 1128 between rim 1137 and ridges 1132 of projections 1130. Spring 1142 is preferably formed of hardened stainless steel.

The positioner 1128 and spring 1142 are operative to move a non-liquid sample up and against electron beam permeable, fluid impermeable, membrane 1110 when enclosure elements 1100 and 1102 and connecting element 1122 are in tight threaded engagement.

Second enclosure element 1102 is preferably formed with a generally central stub 1150, which is arranged to be seated in a suitable recess (not shown) in a specimen stage of a scanning electron microscope. It is a particular feature of the present invention that the container, shown in Figs. 31A-40, is sized and operative with conventional stub recesses in conventional scanning electron microscopes and does not require any modification thereof whatsoever. It is appreciated that various configurations and sizes of stubs may be provided so as to fit various scanning electron microscopes.

Enclosure elements 1100 and 1102 are preferably also provided with respective radially extending positioning and retaining protrusions 1154 and 1155, to enable the container to be readily seated in a suitable multi-container holder and also to assist users in threadably opening and closing the enclosure elements 1100 and 1102.

Preferably, the mutual azimuthal positioning of the protrusions 1154 and 1155 on respective enclosure elements 1100 and 1102 is such that mutual azimuthal alignment therebetween indicates a desired degree of threaded closure therebetween, as shown in Figs. 34A and 34B.

Reference is now made to Figs. 36A, 36B & 36C, which are three sectional illustrations showing the operative orientation of the SEM compatible sample container of Figs. 31A-35B at three stages of operation. Fig. 36A shows the container of Figs. 31A-35B containing a tissue sample 1160 and arranged in the orientation shown in Fig. 31B, prior to threaded closure of enclosure elements 1100 and 1102 and connecting element 1122. The electron beam permeable, fluid impermeable, membrane 1110 is seen in Fig. 36A to be generally planar.

Fig. 36B shows the container of Fig. 36A immediately following full threaded engagement between enclosure elements 1100 and 1102 and connecting

element 1122 producing sealing of the tissue sample enclosure 1116 from the ambient.

It is noted that the tissue sample 1160 is in close contact with the electron beam permeable, fluid impermeable, membrane 1110 due to the force exerted by the positioner 1128. It is seen that the electron beam permeable, fluid impermeable, membrane 1110 and its supporting grid 1112 bow outwardly due to pressure buildup in the tissue sample enclosure 1116 as the result of sealing thereof in this manner.

Fig. 36C illustrates the container of Fig. 36B, when placed in an evacuated environment of a SEM, typically at a vacuum of 10-2 10-6 millibars. It is seen that in this environment, the electron beam permeable, fluid impermeable, membrane 1110 and support grid 1112 bow outwardly to a greater extent than in the ambient environment of Fig. 36B and further that the electron beam permeable, fluid impermeable, membrane 1110 tends to be forced into and through the interstices of grid 1112 to a greater extent than occurs in the ambient environment of Fig. 36B.

Reference is now made to Fig. 37, which is a simplified sectional and pictorial illustration of tissue containing sample and insertion into a SEM using the SEM compatible sample container of Figs. 31A-36C.

Fig. 37 shows the closed container, in the orientation of Fig. 31B, being inserted onto a stage 1164 of a SEM 1166. It is appreciated that there exist SEMs wherein the orientation of the container is opposite to that shown in Fig. 37.

Reference is now made to Figs. 38A, 38B, 38C and 38D, which are four sectional illustrations showing the operative orientation of a variation of the SEM compatible sample container of Figs. 31A-35B at four stages of operation. Fig. 38A shows a container 1170, identical to the container of Figs. 31A-35B other than as specified hereinbelow, containing a sample including cells 1172 grown on a cell growth platform 1174 and arranged in the orientation shown in Fig. 31B, prior to threaded closure of enclosure elements 1100 and 1102 and connecting element 1122. The electron beam permeable, fluid impermeable, membrane 1110 is seen in Fig. 38A to be generally planar. Cell growth platform 1174 is removably mounted onto a suitably configured positioner 1176, which corresponds to positioner 1128 in the embodiment of Figs. 31A-37. Typically, the cells are grown onto cell growth platform 1174 while platform 1174 is not mounted onto positioner 1176. The mounting of platform 1174 onto positioner 1176 typically occurs just before SEM inspection takes place.

Fig. 38B shows the container of Fig. 38A immediately following full threaded engagement between enclosure elements 1100 and 1102 and connecting element 1122 producing sealing of the cell sample enclosure, here designated by reference numeral 1178, from the ambient. It is noted that the sample containing cells 1172 is in close contact with the electron beam permeable, fluid impermeable, membrane 1110 due to the force exerted by the positioner 1176. It is seen that the electron beam permeable, fluid impermeable, membrane 1110 and its supporting grid 1112 bow outwardly due to pressure buildup in the cell sample enclosure 1178 as the result of sealing thereof in this manner.

Fig. 38C illustrates the container of Fig. 38B, when placed in an evacuated environment of a SEM, typically at a vacuum of 10-2-10-6 millibars. It is seen that in this environment, the electron beam permeable, fluid impermeable, membrane 1110 and support grid 1112 bow outwardly to a greater extent than in the ambient environment of Fig. 38B and further that the electron beam permeable, fluid impermeable, membrane 1110 tends to be forced into and through the interstices of grid 1112 to a greater extent than occurs in the ambient environment of Fig. 38B.

Fig. 38D shows the closed container 1170, in the orientation of Fig. 31B, being inserted onto stage 1164 of SEM 1166. It is appreciated that there exist SEMs wherein the orientation of the container is opposite to that shown in Fig. 38D.

Reference is now made to Fig. 39, which is a simplified pictorial and sectional illustration of SEM inspection of a sample using the SEM compatible sample container of Figs. 31A-37. As seen in Fig. 39, the container, here designated by reference numeral 1180, is shown positioned on stage 1164 of SEM 1166 such that an electron beam 1182, generated by the SEM, passes through electron beam permeable, fluid impermeable, membrane 1110 and impinges on a tissue containing sample 1184 within container 1180. Backscattered electrons from sample 1184 pass through electron beam permeable, fluid impermeable, membrane 1110 and are detected by a detector 1186, forming part of the SEM. One or more additional detectors, such as a secondary electron detector 1188, may also be provided. An X-ray detector (not shown) may also be provided for detecting X-ray radiation emitted by the sample 1184 due to electron beam excitation thereof.

Reference is now made additionally to Fig. 40, which schematically illustrates some details of the electron beam interaction with the sample 1184 in container 1180 in accordance with a preferred embodiment of the present invention. It is noted that the present invention enables high contrast imaging of features which are distinguished from each other by their average atomic number, as illustrated in Fig. 40.

In Fig. 40 it is seen that nucleoli 1190, having a relatively high average atomic number, backscatter electrons more than the surrounding nucleoplasm 1192.

It is also noted that in accordance with a preferred embodiment of the present invention, imaging of the interior of the sample to a depth of up to approximately 2 microns is achievable for electrons having an energy level of less than 50KeV, as seen in Fig. 40, wherein nucleoli 1190 disposed below electron beam permeable, fluid impermeable, membrane 1110 are imaged.

Reference is now made to Figs. 41A-45B, which are oppositely facing simplified exploded view pictorial illustrations of a disassembled scanning electron microscope (SEM) compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention. As seen in Figs. 41 A & 41B, the SEM compatible sample container comprises first and second threaded enclosure elements, respectively designated by reference numerals 1200 and 1202, arranged for enhanced ease and speed of closure. Enclosure elements 1200 and 1202 are preferably molded of plastic and coated with a conductive metal coating.

First enclosure element 1200 preferably defines a sample enclosure and has a base surface 1204 having a generally central aperture 1206. An electron beam permeable, fluid impermeable, membrane subassembly 1208, shown in detail in Figs.

42A and 42B, is seated inside enclosure element 1200 against and over aperture 1206, as shown in Figs. 43A & 43B and 45A & 45B. A sample dish comprising subassembly 1208 suitably positioned in enclosure element 1200 is designated by reference numeral 1209, as shown in Figs. 43A-45B.

Turning additionally to Figs. 42A and 42B, it is seen that an electron beam permeable, fluid impermeable, membrane 1210, preferably a polyimide membrane, such as Catalog No. LWN00033, commercially available from Moxtek Inc. of Orem, UT, U. S. A. , is adhered, as by an adhesive, to a mechanically supporting grid 1212. Grid 1212, which is not shown to scale, is preferably Catalog No. BM 0090-01,

commercially available from Buckbee-Mears of Cortland, N. Y. , U. S. A. , and the adhesive is preferably Catalog No. NOA61, commercially available from Norland Products Inc. of Cranbury, NJ, U. S. A. A sample enclosure defining ring 1214 is adhered to electron beam permeable, fluid impermeable, membrane 1210, preferably by an adhesive, such as Catalog No. NOA61, commercially available from Norland Products Inc. of Cranbury, NJ, U. S. A. Ring 1214 is preferably formed of PMMA (polymethyl methacrylate), such as Catalog No. 692106001000, commercially available from Irpen of Barcelona, Spain, and preferably defines a sample enclosure with a volume of approximately 20 microliters and a height of approximately 2 mm. Preferably ring 1214 is configured to define a sample enclosure 1216 having inclined walls.

A diaphragm 1218 is preferably integrally formed of an O-ring portion 1220 to which is sealed an expandable sheet portion 1221. The diaphragm 1218 is preferably disposed between an interior surface 1219 of second enclosure element 1202 and a connecting element 1222. Connecting element 1222 is preferably molded of plastic and coated with a conductive metal coating. The diaphragm 1218 is preferably molded of silicon rubber having a Shore hardness of about 50 and the sheet portion 1221 preferably has a thickness of 0.2-0. 3 mm.

An 0-ring 1223 is preferably disposed between connecting element 1222 and ring 1214 of subassembly 1208. Diaphragm 1218 and O-ring 1223 are operative, when enclosure elements 1200 and 1202 and connecting element 1222 are in tight threaded engagement, to obviate the need for the threaded engagement of elements 1200 and 1202 and connecting element 1222 to be a sealed engagement and to provide dynamic and static pressure relief.

Connecting element 1222 preferably has a central recess 1224.

Connecting element 1222 is also formed with a protrusion 1226, seen in Figs. 45A& 45B, protruding into recess 1224.

A positioner 1228 is preferably comprised of two upright flexible projections 1230, each with a ridge 1232 formed on an end 1234 of the projections 1230. Positioner 1128 is preferably molded of plastic. Projections 1230 press against each other when inserted into recess 1224 of connecting element 1222 and then snap back to an upright position once ridges 1232 are seated on the protrusion 1226 of connecting element 1222, as shown in Figs. 45A & 45B.

Positioner 1228 is preferably also provided with respective radially extending positioning and retaining protrusions 1236 extending from a rim 1237.

Positioning and retaining protrusions 1236 are seated in apertures 1240 formed in inclined walls of sample enclosure 1216 of ring 1214 to prevent rotation of positioner 1228.

A coil spring 1242 is disposed on positioner 1228 between rim 1237 and ridges 1232 of projections 1230. Spring 1242 is preferably formed of hardened stainless steel.

The positioner 1228 and spring 1242 are operative to move a non-liquid sample up and against electron beam permeable, fluid impermeable, membrane 1210 when enclosure elements 1200 and 1202 and connecting element 1222 are in tight threaded engagement.

Second enclosure element 1202 is preferably formed with a generally central stub 1250, having a throughgoing bore 1252, which stub is arranged to be seated in a suitable recess (not shown) in a specimen stage of a scanning electron microscope.

Bore 1252 enables diaphragm 1218 to provide pressure relief by defining a fluid communication channel between one side of the diaphragm 1218 and the environment in which the (SEM) compatible sample container is located. It is a particular feature of the present invention that the container, shown in Figs. 41A-50, is sized and operative with conventional stub recesses in conventional scanning electron microscopes and does not require any modification thereof whatsoever. It is appreciated that various configurations and sizes of stubs may be provided so as to fit various scanning electron microscopes.

Enclosure elements 1200 and 1202 are preferably also provided with respective radially extending positioning and retaining protrusions 1254 and 1255, to enable the container to be readily seated in a suitable multi-container holder and also to assist users in threadably opening and closing the enclosure elements 1200 and 1202.

Preferably, the mutual azimuthal positioning of the protrusions 1254 and 1255 on respective enclosure elements 1200 and 1202 is such that mutual azimuthal alignment therebetween indicates a desired degree of threaded closure therebetween, as shown in Figs. 44A and 44B.

Reference is now made to Figs. 46A, 46B & 46C, which are three sectional illustrations showing the operative orientation of the SEM compatible sample container of Figs. 41A-45B at three stages of operation. Fig. 46A shows the container of Figs. 41A-45B containing a tissue sample 1260 and arranged in the orientation shown in Fig. 41B, prior to threaded closure enclosure elements 1200 and 1202 and connecting element 1222. The electron beam permeable, fluid impermeable, membrane 1210 is seen in Fig. 46A to be generally planar.

Fig. 46B shows the container of Fig. 46A immediately following full threaded engagement between enclosure elements 1200 and 1202 and connecting element 1222, producing sealing of the tissue sample enclosure 1216 from the ambient.

It is noted that the tissue sample 1260 is in close contact with the electron beam permeable, fluid impermeable, membrane 1210 due to the force exerted by the positioner 1228. It is seen that the diaphragm 1218 bows outwardly due to pressure buildup in the tissue sample enclosure 1216 as the result of sealing thereof in this manner. In this embodiment, electron beam permeable, fluid impermeable, membrane 1210 and its supporting grid 1212 also bow outwardly due to pressure buildup in the tissue sample enclosure 1216 as the result of sealing thereof in this manner, however to a significantly lesser extent, due to the action of diaphragm 1218. This can be seen by comparing Fig. 46B with Fig. 36B.

Fig. 46C illustrates the container of Fig. 46B, when placed in an evacuated environment of a SEM, typically at a vacuum of 10-2-10-6 millibars. It is seen that in this environment, the diaphragm 1218 bows outwardly to a greater extent than in the ambient environment of Fig. 46B and that electron beam permeable, fluid impermeable, membrane 1210 and support grid 1212 also bow outwardly to a greater extent than in the ambient environment of Fig. 46B, but to a significantly lesser extent than in the embodiment of Fig. 36C, due to the action of diaphragm 1218. This can be seen by comparing Fig. 46C with Fig. 36C.

It is also noted that the electron beam permeable, fluid impermeable, membrane 1210 tends to be forced into and through the interstices of grid 1212 to a greater extent than occurs in the ambient environment of Fig. 46B but to a significantly lesser extent than in the embodiment of Fig. 36C, due to the action of diaphragm 1218.

This can also be seen by comparing Fig. 46C with Fig. 36C.

Reference is now made to Fig. 47, which is a simplified sectional and pictorial illustration of tissue containing samples and insertion into a SEM using the SEM compatible sample container of Figs. 41A-46C.

Fig. 47 shows the closed container, in the orientation of Fig. 41B, being inserted onto a stage 1264 of a SEM 1266. It is appreciated that there exist SEMs wherein the orientation of the container is opposite to that shown in Fig. 47.

Reference is now made to Figs. 48A, 48B, 48C and 48D, which are four sectional illustrations showing the operative orientation of a variation of the SEM compatible sample container of Figs. 41A-45B at four stages of operation. Fig. 48A shows a container 1270, identical to the container of Figs. 41A-45B other than as specified hereinbelow, containing a sample including cells 1272 grown on a cell growth platform 1274 and arranged in the orientation shown in Fig. 41B, prior to threaded closure of enclosure elements 1200 and 1202 and connecting element 1222. The electron beam permeable, fluid impermeable, membrane 1210 is seen in Fig. 48A to be generally planar. Cell growth platform 1274 is removably mounted onto a suitably configured positioner 1276, which corresponds to positioner 1228 in the embodiment of Figs. 41A-47. Typically, the cells are grown onto cell growth platform 1274 while platform 1274 is not mounted onto positioner 1276. The mounting of platform 1274 onto positioner 1276 typically occurs just before SEM inspection takes place.

Fig. 48B shows the container of Fig. 48A immediately following full threaded engagement between enclosure elements 1200 and 1202 and connecting element 1222 producing sealing of the cell sample enclosure, here designated by reference numeral 1278, from the ambient. It is noted that the sample containing cells 1272 is in close contact with the electron beam permeable, fluid impermeable, membrane 1210 due to the force exerted by the positioner 1276. It is seen that the electron beam permeable, fluid impermeable, membrane 1210 and its supporting grid 1212 bow outwardly due to pressure buildup in the cell sample enclosure 1278 as the result of sealing thereof in this manner, however to a significantly lesser extent than in the embodiment of Fig. 38B, due to the action of diaphragm 1218. This can be seen by comparing Fig. 48B with Fig. 38B.

Fig. 48C illustrates the container of Fig. 48B, when placed in an evacuated environment of a SEM, typically at a vacuum of 10-2 10-6 millibars. It is

seen that in this environment, the electron beam permeable, fluid impermeable, membrane 1210 and support grid 1212 bow outwardly to a greater extent than in the ambient environment of Fig. 48B and further that the electron beam permeable, fluid impermeable, membrane 1210 tends to be forced into and through the interstices of grid 1212 to a greater extent than occurs in the ambient environment of Fig. 48B, but to a significantly lesser extent than in the embodiment of Fig. 38C, due to the action of diaphragm 1218. This can be seen by comparing Fig. 48C with Fig. 38C.

Fig. 48D shows the closed container 1270, in the orientation of Fig. 41B, being inserted onto stage 1264 of SEM 1266. It is appreciated that there exist SEMs wherein the orientation of the container is opposite to that shown in Fig. 48D.

Reference is now made to Fig. 49, which is a simplified pictorial and sectional illustration of SEM inspection of a sample using the SEM compatible sample container of Figs. 41A-47. As seen in Fig. 49, the container, here designated by reference numeral 1280, is shown positioned on stage 1264 of SEM 1266 such that an electron beam 1282, generated by the SEM, passes through electron beam permeable, fluid impermeable, membrane 1210 and impinges on a tissue containing sample 1284 within container 1280. Backscattered electrons from sample 1284 pass through electron beam permeable, fluid impermeable, membrane 1210 and are detected by a detector 1286, forming part of the SEM. One or more additional detectors, such as a secondary electron detector 1288, may also be provided. An X-ray detector (not shown) may also be provided for detecting X-ray radiation emitted by the sample 1284 due to electron beam excitation thereof.

Reference is now made additionally to Fig. 50, which schematically illustrates some details of the electron beam interaction with the sample 1284 in container 1280 in accordance with a preferred embodiment of the present invention. It is noted that the present invention enables high contrast imaging of features which are distinguished from each other by their average atomic number, as illustrated in Fig. 50.

In Fig. 50 it is seen that nucleoli 1290, having a relatively high average atomic number, backscatter electrons more than the surrounding nucleoplasm 1292.

It is also noted that in accordance with a preferred embodiment of the present invention, imaging of the interior of the sample to a depth of up to approximately 2 microns is achievable for electrons having an energy level of less than

50KeV, as seen in Fig. 50, wherein nucleoli 1290 disposed below electron beam permeable, fluid impermeable, membrane 1210 are imaged.

Reference is now made to Figs. 51A, 51B and 51C, which are simplified illustrations of a microscopy multi-sample holder in use with a SEM compatible sample dish of the type shown in Figs. 31A-40. As seen in Fig. 51A, the microscopy multi-sample holder preferably comprises a base 1400 and a sealing cover 1404. The base 1400 is preferably injection molded of a plastic material and defines an array of dish support locations 1406. Each dish support location 1406 is preferably defined by an aperture 1408 through which SEM microscopy may take place. Adjacent to each aperture 1408 there is preferably formed a pair of mutually aligned pairs of upstanding mutually spaced protrusions 1410 arranged to receive protrusions 1424 on sample dishes 1425. Sample dishes 1425 may be generally identical to sample dishes 1109, shown in Figs. 33A-35B, but do not require any threading or other attachment mechanism.

Sealing cover 1404 is preferably arranged for individual sealing engagement with each of sample dishes 1425. Preferably sealing cover 1404 is provided on the underside thereof with an array of 0-rings 1426, shown in Fig 51C, sealed thereto and arranged so as to sealingly engage a top rim surface of each of sample dishes 1425, when the sealing cover 1404 is in place, preferably in removable snap-fit engagement with base 1400.

Preferably, sealing cover 1404 is provided on the underside thereof with an array of positioners 1428, shown in Fig. 51C, and arranged so as to move non-liquid samples up and against electron beam permeable, fluid impermeable, membrane 1110 (shown in Figs. 31A-40) seated in sample dish 1425. Individual positioners 1428 are suspended within coils 1430, as shown in Fig. 51C.

Fig. 51B shows the apparatus of Fig. 51A with one sample dish 1425 positioned at a dish support location 1406 in base 1400. Fig. 51C shows sealing cover 1404 in snap fit engagement with base 1400, thereby providing individual sealing of each of sample dishes 1425 by means of O-ring 1426 and a portion of sealing cover 1404 circumscribed thereby.

Reference is now made to Figs. 52A, 52B and 52C, which are simplified illustrations of a microscopy multi-sample holder in use with a SEM compatible sample

dish of the type shown in Figs. 41A-50. As seen in Fig. 52A, the microscopy multi-sample holder preferably comprises a base 1450 and a sealing cover 1454. The base 1450 is preferably injection molded of a plastic material and defines an array of dish support locations 1456. Each dish support location 1456 is preferably defined by an aperture 1458 through which SEM microscopy may take place. Adjacent to each aperture 1458 there is preferably formed a pair of mutually aligned pairs of upstanding mutually spaced protrusions 1460 arranged to receive protrusions 1474 on sample dishes 1475. Sample dishes 1475 may be generally identical to sample dishes 1209, shown in Figs. 43A-45B, but do not require any threading or other attachment mechanism.

Preferably sealing cover 1454 is provided on the underside thereof with an array of 0-rings 1476, shown in Fig 52C, sealed thereto and arranged so as to sealingly engage a top rim surface of each of sample dishes 1475, when the sealing cover 1454 is in place, preferably in removable snap-fit engagement with base 1450.

Sealing cover 1454 is arranged for individual sealing engagement of each of sample dishes 1475 with a diaphragm 1477, shown in Fig. 52C, which is sealingly mounted over an aperture 1478 formed in sealing cover 1454. Preferably an array of diaphragms 1477, which may be identical to diaphragms 1218 described hereinabove with reference to Figs. 41A-50, is provided on the underside of sealing cover 1454.

The individual diaphragms 1477 are arranged so as to sealingly engage a top rim surface of each of sample dishes 1475, when the sealing cover 1454 is in place, preferably in removable snap-fit engagement with base 1450.

Preferably, sealing cover 1454 is provided with an array of positioners 1480. Individual positioners 1480 are suspended within coils 1482, as shown in Fig. 51C, so as to move non-liquid samples up and against electron beam permeable, fluid impermeable, membrane 1210 (shown in Figs. 41A-50) seated in sample dish 1475.

Fig. 52B shows the apparatus of Fig. 52A with one sample dish 1475 positioned at a dish support location 1456 in base 1450. Fig. 52C shows sealing cover 1454 in snap fit engagement with base 1450, thereby providing individual sealing of each of sample dishes 1475 by means of diaphragm 1476.

Reference is now made to Figs. 53A and 53B, which are simplified illustrations of a microscopy multi-sample holder defining a plurality of SEM compatible sample containers in accordance with a preferred embodiment of the present invention. As seen in Fig. 53A, the microscopy multi-sample holder preferably comprises a base 1500 and a sealing cover 1504. The base 1500 is preferably injection molded of a plastic material and defines an array of sample containers 1506. Each sample container 1506 preferably includes an aperture 1508 through which SEM microscopy may take place. An electron beam permeable, fluid impermeable, membrane 1510, shown in Fig. 53B, is sealed over each aperture 1508. Membrane 1510 is preferably identical to membrane 1110 described hereinabove with reference to Figs.

31A-40. Sealing cover 1504 preferably is arranged for individual sealing engagement with each of sample containers 1506.

Preferably, sealing cover 1504 is provided with an array of positioners 1520, shown in Fig. 53B. Individual positioners 1520 are suspended within coils 1522, as shown in Fig. 53B, so as to move non-liquid samples up and against electron beam permeable, fluid impermeable, membrane 1510.

Fig. 53B shows the apparatus of Fig. 53A in sealed engagement, thereby providing individual sealing of each of sample containers 1506.

Reference is now made to Figs. 54A and 54B, which are simplified illustrations of a microscopy multi-sample holder defining a plurality of SEM compatible sample containers in accordance with a preferred embodiment of the present invention. As seen in Fig. 54A, the microscopy multi-sample holder preferably comprises a base 1550 and a sealing cover 1554. The base 1550 is preferably injection molded of a plastic material and defines an array of sample containers 1556. Each sample container 1556 preferably includes an aperture 1558 through which SEM microscopy may take place.

An electron beam permeable, fluid impermeable, membrane 1560, shown in Fig. 54B, is sealed over each aperture 1558. Membrane 1560 is preferably identical to membrane 1210 described hereinabove with reference to Figs. 41A-50. Sealing cover 1554 preferably comprises a diaphragm 1562 formed of resilient sheet material such as silicon rubber of 0.2-0. 3 mm in thickness and having a Shore hardness of about 50.

Diaphragm 1562 is sealingly mounted over apertures 1564 formed in sealing cover 1554 and is arranged for individual sealing engagement with each of sample containers 1556.

Preferably, sealing cover 1554 is provided with an array of positioners 1570, shown in Fig. 54B. Individual positioners 1570 are suspended within coils 1572, as shown in Fig. 54B, so as to move non-liquid samples up and against electron beam permeable, fluid impermeable, membrane 1560.

Fig. 54B shows the apparatus of Fig. 54A in sealed engagement, thereby providing individual sealing of each of sample containers 1556.

Reference is now made to Fig. 55, which is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with a preferred embodiment of the present invention. As seen in Fig. 55, preferably, automated positioning systems, such as robotic arms, as shown, are used for conveying a multiplicity of SEM compatible sample containers 1602 throughout the system, it being appreciated that manual intervention may be employed at one or more stages as appropriate.

Thereafter, individual containers 1602 are placed on a removable electron microscope specimen stage 1610, which is subsequently introduced into a scanning electron microscope 1612. The resulting image may be inspected visually by an operator and/or analyzed by conventional image analysis functionality, typically embodied in a computer 1614.

Reference is now made to Fig. 56, which is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with another preferred embodiment of the present invention. As seen in Fig. 56, a plurality of microscopy multi-sample holders 1650, each containing a multiplicity of SEM compatible sample dishes 1652 of either of the types shown in Figs. 51 A-52C, is shown on a table 1654. Preferably, light microscopy inspection of the samples in sample dishes 1652 is carried out while the sample dishes are mounted in holder 1650, as indicated at reference numeral 1656, in order to identify samples of interest. Preferably a dissection microscope 1658 is employed for this purpose.

Preferably automated positioning systems, such as robotic arms, as shown, are used for conveying the microscopy multi-sample holders 1650 containing

sample dishes 1652 throughout the system, it being appreciated that manual intervention may be employed at one or more stages as appropriate.

Thereafter, holders 1650 are placed on an electron microscope specimen stage 1660, which is subsequently introduced into a scanning electron microscope 1662.

The resulting images may be inspected visually by an operator and/or analyzed by conventional image analysis functionality, typically embodied in a computer 1664.

Reference is now made to Fig. 57, which is a simplified illustration of a SEM based sample inspection system constructed and operative in accordance with yet another preferred embodiment of the present invention. As seen in Fig. 57, a plurality of microscopy multi-sample holders 1670, each defining a multiplicity of SEM compatible sample containers 1672, as shown in any of Figs. 53A-54B, are seen on a table 1674.

Preferably, light microscopy inspection of the samples in sample containers 1672 is carried out holder-wise, as indicated at reference numeral 1676, in order to identify samples of interest. Preferably a dissection microscope 1678 is employed for this purpose.

Preferably automated positioning systems, such as robotic arms, as shown, are used for conveying the microscopy multi-sample holders 1670 throughout the system, it being appreciated that manual intervention may be employed at one or more stages as appropriate.

Thereafter, holders 1670 are placed on an electron microscope specimen stage 1680, which is subsequently introduced into a scanning electron microscope 1682.

The resulting images may be inspected visually by an operator and/or analyzed by conventional image analysis functionality, typically embodied in a computer 1684.

Reference is now made to Figs. 58A-62B, which are oppositely facing simplified exploded view pictorial illustrations of a disassembled scanning electron microscope (SEM) compatible sample container constructed and operative in accordance with yet another preferred embodiment of the present invention. As seen in Figs. 58A & 58B, the SEM compatible sample container comprises first and second mutually threaded enclosure elements, respectively designated by reference numerals 2100 and 2102, arranged for enhanced ease and speed of closure. Enclosure elements 2100 and 2102 are preferably molded of plastic and coated with a conductive metal coating.

First enclosure element 2100 preferably defines a liquid sample enclosure and has a base surface 2104 having a generally central aperture 2106. An electron beam permeable, fluid impermeable, membrane subassembly 2108, shown in detail in Figs.

59A and 59B, is seated inside enclosure element 2100 against and over aperture 2106, as shown in Figs. 60A & 60B and 62A & 62B. A sample dish comprising subassembly 2108 suitably positioned in enclosure element 2100 is designated by reference numeral 2109, as shown in Figs. 60A-62B.

Turning additionally to Figs. 59A and 59B, it is seen that an electron beam permeable, fluid impermeable, membrane 2110, preferably a polyimide membrane, such as Catalog No. LWN00033, commercially available from Moxtek Inc. of Orem, UT, U. S. A. , is adhered, as by an adhesive, to a mechanically supporting grid 2112. Grid 2112, which is not shown to scale, is preferably Catalog No. BM 0090-01, <BR> <BR> commercially available from Buckbee-Mears of Cortland, N. Y. , U. S. A. , and the adhesive is preferably Catalog No. NOA61, commercially available from Norland Products Inc. of Cranbury, NJ, U. S. A. A liquid sample enclosure defining ring 2114 is adhered to electron beam permeable, fluid impermeable, membrane 2110, preferably by an adhesive, such as Catalog No. NOA61, commercially available from Norland Products Inc. of Cranbury, NJ, U. S. A. Ring 2114 is preferably formed of PMMA (polymethyl methacrylate), such as Catalog No. 692106001000, commercially available from Irpen of Barcelona, Spain, and preferably defines a liquid sample enclosure with a volume of approximately 20 microliters and a height of approximately 2 mm. Preferably ring 2114 is configured to define a liquid sample enclosure 2116 having inclined walls.

An O-ring 2118 is preferably disposed between ring 2114 and an interior surface 2120 of second enclosure element 2102. O-ring 2118 is operative, when enclosure elements 2100 and 2102 are in tight threaded engagement, to obviate the need for the threaded engagement of elements 2100 and 2102 to be a sealed engagement.

Second enclosure element 2102 preferably is formed with a generally central stub 2122, having a throughgoing bore 2123, which stub is arranged to be seated in a suitable recess (not shown) in a specimen stage of a scanning electron microscope.

Enclosure elements 2100 and 2102 are preferably also provided with respective radially extending positioning and retaining protrusions 2124 and 2125, to enable the container to be readily seated in a suitable multi-container holder and also to

assist users in threadably opening and closing the enclosure elements 2100 and 2102.

Preferably, the mutual azimuthal positioning of the protrusions 2124 and 2125 on respective enclosure elements 2100 and 2102 is such that mutual azimuthal alignment therebetween indicates a desired degree of threaded closure therebetween, as shown in Figs. 61A and 61B.

A light guide 2126 is provided to receive light from a sample in liquid sample enclosure 2116 during SEM inspection from a side of the sample not facing the electron beam permeable, fluid impermeable, membrane 2110.

The light guide 2126 is formed of a cylinder with a truncated conical tip 2128 and is inserted into bore 2123 of stub 2122. Light guide 2126 is preferably a plastic or glass clad light guide with a numerical aperture of 0.66, commercially <BR> <BR> available from Fiberoptics Technology, Inc. of 12 Fiber Rd. , Pomfret, CT, U. S. A. and is sealed to enclosure 2102 by any conventional means, such as by an adhesive.

The wall of conical tip 2128 is configured at an angle, such that the angle is smaller than a critical angle of reflection, so as to ensure that incident photons emitted from a sample are reflected in the light guide 2126.

Reference is now made to Figs. 63A, 63B & 63C, which are three sectional illustrations showing the operative orientation of the SEM compatible sample container of Figs. 58A-62B at three stages of operation. Fig. 63A shows the container of Figs. 58A-62B containing a liquid sample 2130 and arranged in the orientation shown in Fig. 58B, prior to threaded closure of enclosure elements 2100 and 2102. It is noted that the liquid sample does not flow out of the liquid sample enclosure 2116 due to surface tension. The electron beam permeable, fluid impermeable, membrane 2110 is seen in Fig. 63A to be generally planar.

Fig. 63B shows the container of Fig. 63A immediately following full threaded engagement between enclosure elements 2100 and 2102, producing sealing of the liquid sample enclosure 2116 from the ambient. The light guide tip 2128 is shown to be immersed in liquid sample 2130. It is seen that the electron beam permeable, fluid impermeable, membrane 2110 and its supporting grid 2112 bow outwardly due to pressure buildup in the liquid sample enclosure 2116 as the result of sealing thereof in this manner.

Fig. 63C illustrates the container of Fig. 63B, when placed in an evacuated environment of a SEM, typically at a vacuum of 10-2-10-6 millibars. It is seen that in this environment, the electron beam permeable, fluid impermeable, membrane 2110 and support grid 2112 bow outwardly to a greater extent than in the ambient environment of Fig. 63B and further that the electron beam permeable, fluid impermeable, membrane 2110 tends to be forced into and through the interstices of grid 2112 to a greater extent than occurs in the ambient environment of Fig. 63B.

Reference is now made to Figs. 64A, 64B, 64C, 64D and 64E, which are simplified sectional illustrations of cell growth, liquid removal, liquid addition, sealing and insertion into a SEM respectively using the SEM compatible sample container of Figs. 58A-63C. Turning to Fig. 64A, which illustrates a typical cell culture situation, it is seen that the enclosure element 2100 having disposed therewithin subassembly 2108 is in the orientation shown in Fig. 58A and cells 2140 in a liquid medium 2142 are located within liquid sample enclosure 2116, the cells 2140 lying against the electron beam permeable, fluid impermeable, membrane 2110.

Fig. 64B shows removal of liquid from liquid sample enclosure 2116, typically by aspiration, and Fig. 64C shows addition of liquid to liquid sample enclosure 2116. It is appreciated that multiple occurrences of liquid removal and addition may take place with respect to a sample within liquid sample enclosure 2116. Preferably, the apparatus employed for liquid removal and addition is designed or equipped such as to prevent inadvertent rupture of the electron beam permeable, fluid impermeable, membrane 2110.

Fig. 64D illustrates closing of the container containing the cells 2140, seen in Fig. 64C, in a liquid medium 2142. The light guide tip 2128 is shown to be immersed in liquid medium 2142 of the liquid sample 2140. Fig. 64E shows the closed container, in the orientation of Fig. 58B being inserted onto a stage 2144 of a SEM 2146. It is appreciated that there exist SEMs wherein the orientation of the container is opposite to that shown in Fig. 64E.

Figs. 64A-64D exemplify a situation wherein at least a portion of a liquid containing sample remains in contact with the electron beam permeable, fluid impermeable, membrane 2110 notwithstanding the addition or removal of liquid from liquid sample enclosure 2116. This situation may include situations wherein part of the

sample is adsorbed or otherwise adhered to the electron beam permeable, fluid impermeable, membrane 2110. Examples of liquid containing samples may include cell cultures, blood, bacteria and acellular material.

Reference is now made to Figs. 65A, 65B and 65C which are simplified sectional illustrations of liquid containing samples in contact with the electron beam permeable, fluid impermeable, membrane 2110, sealing and insertion into a SEM respectively, using the SEM compatible sample container of Figs. 58A-63C. Figs. 65A - 65C exemplify a situation wherein at least a portion of a liquid containing sample 2160 is in contact with the electron beam permeable, fluid impermeable, membrane 2110 but is not adhered thereto. Examples of liquid containing samples may include various emulsions and suspensions such as milk, cosmetic creams, paints, inks and pharmaceuticals in liquid form. It is seen that the enclosure element 2100 in Figs. 65A and 65B, having disposed therewithin subassembly 2108, is in the orientation shown in Fig. 58A.

Fig. 65B illustrates closing of the container containing the sample 2160.

Fig. 65C shows the closed container, in the orientation of Fig. 58B, being inserted onto stage 2144 of SEM 2146. It is appreciated that there exist SEMs wherein the orientation of the container is opposite to that shown in Fig. 65C.

Reference is now made to Fig. 66, which is a simplified pictorial and sectional illustration of SEM inspection with light detection of a sample using the SEM compatible sample container of Figs. 58A-63C. As seen in Fig. 66, the container, here designated by reference numeral 2170, is shown positioned on stage 2144 of SEM 2146 in a recess 2171. Stage 2144 is operative to rotate so as to enable positioning of container 2170 under an electron beam 2172 to inspect, during SEM inspection, regions of interest within a liquid containing sample 2174.

The electron beam 2172, generated by the SEM, passes through electron beam permeable, fluid impermeable, membrane 2110 and impinges on sample 2174 within container 2170. Backscattered electrons from sample 2174 pass through electron beam permeable, fluid impermeable, membrane 2110 and are detected by a detector 2176, forming part of the SEM. One or more additional detectors, such as a secondary electron detector 2178, may also be provided. An X-ray detector (not shown) may also

be provided for detecting X-ray radiation emitted by the sample 2174 due to electron beam excitation thereof.

Photons 2180, emitted from liquid sample 2174 due to electron beam excitation, are transmitted through a first light guide, here designated by reference numeral 2184, which is the same as light guide 2126 of Figs. 58A-63C, to a second light guide 2186. Second light guide 2186 is operative to transmit the photons 2180 to a light detector, such as a Photomultiplier Tube (PMT) 2188. Time dependent measurement of light intensity obtained from the light detector 2188, combined with information on the location of the scanning electron beam, are combined to produce an image of sample 2174 by methods known in the art, preferably as a digital image on a computer 2189.

Second light guide 2186, preferably, has a cross section with a diameter that is larger than the diameter of the cross section of the first light guide 2184 so as to minimize loss of photons in the passage between light guides 2184 and 2186 due to refraction or to imprecise relative alignment of the two light guides.

In the illustrated embodiment, second light guide 2186 is preferably formed in an L-shaped curve and preferably comprises a multiplicity of optic fibers disposed along the L-shaped light guide 2186 at an angle that ensures internal reflection of photons 2180 throughout the length of second light guide 2186.

It is appreciated that in the present invention photons 2180 may be transmitted vertically downward from first light guide 2184, via second light guide 2186 to a light detector located beneath the floor of the SEM 2146. Alternatively, photons 2180 may be transmitted from first light guide 2184 to a light detector, and that second light guide 2186 may be obviated.

In another embodiment of the present invention, the photons 2180 may be spectrally resolved prior to detection by light detector 2188 by conventional means, such as filters, diffraction gratings or prisms (not shown). This allows detection of photons with a wavelength within one or more specified ranges, yielding additional information on the composition and structure of the sample 2174 or features within sample 2174.

Additionally, a liquid, such as oil or a gel (not shown), with an index of refraction similar to the index of refraction of light guides 2184 and 2186, may be

placed between light guides 2184 and 2186 to prevent the photons 2180 from scattering outside light guides 2184 and 2186.

Reference is now made additionally to Fig. 67, which schematically illustrates some details of the electron beam and photon interaction with the sample 2174 in container 2170 in accordance with a preferred embodiment of the present invention. It is noted that the present invention enables high contrast imaging of features which are distinguished from each other by their average atomic number or, alternatively, by their average photon yield due to excitation by electrons, as illustrated in Fig. 67. In Fig. 67 it is seen that nucleoli 2190, having a relatively high average atomic number, backscatter electrons more than the surrounding nucleoplasm 2192.

Photons 2180 are shown to emit from the nucleoli 2190 and are transmitted to the light detector 2188 (shown in Fig. 66), via light guide 2184. It is noted that the contrast obtained by detection of backscattered electrons and the contrast obtained by photon detection are due to different physical processes and to different chemical properties of features within the sample, and therefore do not generally overlap.

It is also noted that in accordance with a preferred embodiment of the present invention, imaging of the interior of the sample to a depth of up to approximately 2 microns is achievable when employing electron beams having an energy level of less than 50KeV, as seen in Fig. 67, wherein nucleoli 2190 disposed below electron beam permeable, fluid impermeable, membrane 2110 are imaged.

Reference is now made to Figs. 68A-72B, which are oppositely facing simplified exploded view pictorial illustrations of a disassembled scanning electron microscope (SEM) compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention. As seen in Figs. 68A & 68B, the SEM compatible sample container comprises first and second mutually threaded enclosure elements, respectively designated by reference numerals 2200 and 2202, arranged for enhanced ease and speed of closure. Enclosure elements 2200 and 2202 are preferably molded of plastic and coated with a conductive metal coating.

First enclosure element 2200 preferably defines a liquid sample enclosure and has a base surface 2204 having a generally central aperture 2206. An electron beam

permeable, fluid impermeable, membrane subassembly 2208, shown in detail in Figs.

69A and 69B, is seated inside enclosure element 2200 against and over aperture 2206, as shown in Figs. 70A & 70B and 72A & 72B. A sample dish comprising subassembly 2208 suitably positioned in enclosure element 2200 is designated by reference numeral 2209, as shown in Figs. 70A-72B.

Turning additionally to Figs. 69A and 69B, it is seen that an electron beam permeable, fluid impermeable, membrane 2210, preferably a polyimide membrane, such as Catalog No. LWN00033, commercially available from Moxtek Inc. of Orem, UT, U. S. A. , is adhered, as by an adhesive, to a mechanically supporting grid 2212. Grid 2212, which is not shown to scale, is preferably Catalog No. BM 0090-01, commercially available from Buckbee-Mears of Cortland, N. Y. , U. S. A. , and the adhesive is preferably Catalog No. NOA61, commercially available from Norland Products Inc. of Cranbury, NJ, U. S. A. A liquid sample enclosure defining ring 2214 is adhered to electron beam permeable, fluid impermeable, membrane 2210, preferably by an adhesive, such as Catalog No. NOA61, commercially available from Norland Products Inc. of Cranbury, NJ, U. S. A. Ring 2214 is preferably formed of PMMA (polymethyl methacrylate), such as Catalog No. 692106001000, commercially available from Irpen of Barcelona, Spain, and preferably defines a liquid sample enclosure with a volume of approximately 20 microliters and a height of approximately 2 mm. Preferably ring 2214 is configured to define a liquid sample enclosure 2216 having inclined walls.

A diaphragm 2218 is preferably provided and is operative to provide dynamic and static pressure relief. Diaphragm 2218 is preferably integrally formed of an O-ring portion 2220 to which is sealed an expandable sheet portion 2221. The diaphragm 2218 is preferably molded of silicon rubber having a Shore hardness of about 50 and the sheet portion 2221 preferably has a thickness of 0.2-0. 3 mm.

The diaphragm 2218 is inserted into a first aperture 2222 formed in an exterior surface of a wall of the second enclosure element 2202. A second aperture 2223, shown in Fig. 72A&72B, is formed in an interior surface of a wall of the second enclosure element 2202. First aperture 2222 and second aperture 2223 enable diaphragm 2218 to provide pressure relief by defining a fluid communication channel between one side of the diaphragm 2218 and the environment in which the SEM compatible sample container is located. A plug 2224 is preferably provided to retain the

diaphragm 2218 in aperture 2222. Plug 2224 is preferably formed of a ring 2225 having a generally central aperture 2226 and is attached to an internal surface of second enclosure element 2202 defined by aperture 2222 by any conventional means, such as by a tight fitting engagement.

An O-ring 2228 is preferably disposed between ring 2214 and an interior surface 2230 of second enclosure element 2202. O-ring 2228 is operative, when enclosure elements 2200 and 2202 are in tight threaded engagement, to obviate the need for the threaded engagement of elements 2200 and 2202 to be a sealed engagement.

Second Enclosure element 2202 preferably is formed with a generally central stub 2232, having a throughgoing bore 2233, which stub is arranged to be seated in a suitable recess (not shown) in a specimen stage of a scanning electron microscope.

Enclosure elements 2200 and 2202 are preferably also provided with respective radially extending positioning and retaining protrusions 2234 and 2235, to enable the container to be readily seated in a suitable multi-container holder and also to assist users in threadably opening and closing the enclosure elements 2200 and 2202.

Preferably, the mutual azimuthal positioning of the protrusions 2234 and 2235 on respective enclosure elements 2200 and 2202 is such that mutual azimuthal alignment therebetween indicates a desired degree of threaded closure therebetween, as shown in Figs. 71A and 71B.

A light guide 2236 is provided to receive light from a sample in liquid sample enclosure 2216 during SEM inspection from a side of the sample not facing the electron beam permeable, fluid impermeable, membrane 2210.

Light guide 2236 is formed of a cylinder with a truncated conical tip 2238 and is inserted into bore 2233 of stub 2232. Light guide 2236 is preferably a plastic or glass clad light guide with a numerical aperture of 0.66, commercially available from Fiberoptics Technology, Inc. of 12 Fiber Rd. , Pomfret, CT, U. S. A and is sealed to enclosure 2202 by any conventional means, such as by an adhesive.

The wall of conical tip 2238 is configured at an angle, such that the angle is smaller than a critical angle of reflection, so as to ensure that incident photons emitted from a sample are reflected in the light guide 2236.

Reference is now made to Figs. 73A, 73B & 73C, which are three sectional illustrations showing the operative orientation of the SEM compatible sample

container of Figs. 68A-72B at three stages of operation. Fig. 73A shows the container of Figs. 68A-72B containing a liquid sample 2239 and arranged in the orientation shown in Fig. 68B, prior to threaded closure of enclosure elements 2200 and 2202. It is noted that the liquid sample does not flow out of the liquid sample enclosure 2216 due to surface tension. The electron beam permeable, fluid impermeable, membrane 2210 is seen in Fig. 73A to be generally planar.

Fig. 73B shows the container of Fig. 73A immediately following full threaded engagement between enclosure elements 2200 and 2202, producing sealing of the liquid sample enclosure 2216 from the ambient. The light guide tip 2238 is shown to be immersed in liquid sample 2239. It is seen that the diaphragm 2218 bows outwardly due to pressure buildup in the liquid sample enclosure 2216 as the result of sealing thereof in this manner. In this embodiment, electron beam permeable, fluid impermeable, membrane 2210 and its supporting grid 2212 also bow outwardly due to pressure buildup in the liquid sample enclosure 2216 as the result of sealing thereof in this manner, however to a significantly lesser extent than in the embodiment of Fig.

63B, due to the action of diaphragm 2218. This can be seen by comparing Fig. 73B with Fig. 63B.

Fig. 73C illustrates the container of Fig. 73B, when placed in an evacuated environment of a SEM, typically at a vacuum of 10'2-10-6 millibars. It is seen that in this environment, the diaphragm 2218 bows outwardly to a greater extent than in the ambient environment of Fig. 73B and that electron beam permeable, fluid impermeable, membrane 2210 and support grid 2212 also bow outwardly to a greater extent than in the ambient environment of Fig. 73B, but to a significantly lesser extent than in the embodiment of Fig. 63C, due to the action of diaphragm 2218. This can be seen by comparing Fig. 73C with Fig. 63C.

It is also noted that the electron beam permeable, fluid impermeable, membrane 2210 tends to be forced into and through the interstices of grid 2212 to a greater extent than occurs in the ambient environment of Fig. 73B but to a significantly lesser extent than in the embodiment of Fig. 63C, due to the action of diaphragm 2218.

This can also be seen by comparing Fig. 73C with Fig. 63C.

Reference is now made to Figs. 74A, 74B, 74C, 74D and 74E, which are simplified sectional illustrations of cell growth, liquid removal, liquid addition, sealing

and insertion into a SEM, respectively, using the SEM compatible sample container of Figs. 68A-73C. Turning to Fig. 74A, which is identical to Fig. 64A and illustrates a typical cell culture situation, it is seen that the enclosure element 2200 having disposed therewithin subassembly 2208 is in the orientation shown in Fig. 68A and cells 2240 in a liquid medium 2242 are located within liquid sample enclosure 2216, the cells 2240 lying against the electron beam permeable, fluid impermeable, membrane 2210.

Fig. 74B, which is identical to Fig. 64B, shows removal of liquid from liquid sample enclosure 2216, typically by aspiration, and Fig. 74C, which is identical to Fig. 64C, shows addition of liquid to liquid sample enclosure 2216. It is appreciated that multiple occurrences of liquid removal and addition may take place with respect to a sample within liquid sample enclosure 2216. Preferably, the apparatus employed for liquid removal and addition is designed or equipped such as to prevent inadvertent rupture of the electron beam permeable, fluid impermeable, membrane 2210.

Fig. 74D illustrates closing of the container containing the cells 2240, seen in Fig. 74C, in a liquid medium 2242. The light guide tip 2238 is shown to be immersed in liquid medium 2242 of the liquid sample. Fig. 74E shows the closed container, in the orientation of Fig. 68B, being inserted onto a stage 2244 of a SEM 2246. It is appreciated that there exist SEMs wherein the orientation of the container is opposite to that shown in Fig. 74E.

Figs. 74A-74D exemplify a situation wherein at least a portion of a liquid containing sample remains in contact with the electron beam permeable, fluid impermeable, membrane 2210 notwithstanding the addition or removal of liquid from liquid sample enclosure 2216. This situation may include situations wherein part of the sample is adsorbed or otherwise adhered to the electron beam permeable, fluid impermeable, membrane 2210. Examples of liquid containing samples may include cell cultures, blood, bacteria and acellular material.

Reference is now made to Figs. 75A, 75B and 75C which are simplified sectional illustrations of liquid containing samples in contact with the electron beam permeable, fluid impermeable, membrane 2210, sealing and insertion into a SEM, respectively, using the SEM compatible sample container of Figs. 68A-73C. Figs. 75A - 75C exemplify a situation wherein at least a portion of a liquid containing sample 2260 is in contact with the electron beam permeable, fluid impermeable, membrane

2210 but is not adhered thereto. Examples of liquid containing samples may include various emulsions and suspensions such as milk, cosmetic creams, paints, inks and pharmaceuticals in liquid form. It is seen that the enclosure element 2200 in Figs. 75A and 75B, having disposed therewithin subassembly 2208, is in the orientation shown in Fig. 68A. Fig. 75A is identical to Fig. 65A.

Fig. 75B illustrates closing of the container containing the sample 2260.

Fig. 75C shows the closed container, in the orientation of Fig. 68B, being inserted onto stage 2244 of SEM 2246. It is appreciated that there exist SEMs wherein the orientation of the container is opposite to that shown in Fig. 75C.

Reference is now made to Fig. 76, which is a simplified pictorial and sectional illustration of SEM inspection with light detection of a sample using the SEM compatible sample container of Figs. 68A-73C. As seen in Fig. 76, the container, here designated by reference numeral 2270, is shown positioned on stage 2244 of SEM 2246 in a recess 2271. Stage 2244 is operative to rotate so as to enable positioning of container 2270 under an electron beam 2272 to inspect, during SEM inspection, regions of interest within a liquid containing sample 2274.

The electron beam 2272, generated by the SEM, passes through electron beam permeable, fluid impermeable, membrane 2210 and impinges on sample 2274 within container 2270. Backscattered electrons from sample 2274 pass through electron beam permeable, fluid impermeable, membrane 2210 and are detected by a detector 2276, forming part of the SEM. One or more additional detectors, such as a secondary electron detector 2278, may also be provided. An X-ray detector (not shown) may also be provided for detecting X-ray radiation emitted by the sample 2274 due to electron beam excitation thereof.

Photons 2280, emitted from liquid sample 2274 due to electron beam excitation, are transmitted through a first light guide, here designated by reference numeral 2284, which is identical to light guide 2226 of Figs. 68A-73C, to a second light guide 2286. Second light guide 2286 is operative to transmit the photons 2280 to a light detector, such as a Photomultiplier Tube (PMT) 2288, such as Catalog No. H6180-01, commercially available at Hamamatsu Photonics of 325-6, Sunayama-cho, Hamamatsu City, Shizuoka Pref. Japan. Time dependent measurement of light intensity obtained from the light detector 2288, combined with information on the location of the scanning

electron beam, are combined to produce an image of sample 2274 by methods known in the art, preferably as a digital image on a computer 2289.

Second light guide 2286, preferably, has a cross section with a diameter that is larger than the diameter of the cross section of the first light guide 2284, so as to minimize loss of photons in the passage between light guides 2284 and 2286 due to refraction or to imprecise relative alignment of the two light guides.

In the illustrated embodiment, second light guide 2286 is preferably formed in an L-shaped curve and preferably comprises a multiplicity of optic fibers disposed along the L-shaped light guide 2286 at an angle that ensures internal reflection of photons 2280 throughout the length of second light guide 2286.

It is appreciated that in the present invention photons 2280 may be transmitted vertically downward from first light guide 2284, via second light guide 2286 to a light detector located beneath the floor of the SEM 2246. Alternatively, photons 2280 may be transmitted from first light guide 2284 to a light detector, and that second light guide 2286 may be obviated.

In another embodiment of the present invention, the photons 2280 may be spectrally resolved prior to detection by light detector 2288 by conventional means, such as filters, diffraction gratings or prisms (not shown). This allows detection of photons with a wavelength within one or more specified ranges, yielding additional information on the composition and structure of the sample 2274 or features within sample 2274.

Additionally, a liquid, such as oil or a gel (not shown), with an index of refraction similar to the index of refraction of light guides 2284 and 2286, may be placed between light guides 2284 and 2286 to prevent the photons 2280 from scattering outside light guides 2284 and 2286.

Reference is now made additionally to Fig. 77, which schematically illustrates some details of the electron beam and photon interaction with the sample 2274 in container 2270 in accordance with a preferred embodiment of the present invention. It is noted that the present invention enables high contrast imaging of features which are distinguished from each other by their average atomic number, or, alternatively, by their average photon yield due to excitation by electrons, as illustrated

in Fig. 77. In Fig. 77 it is seen that nucleoli 2290, having a relatively high average atomic number, backscatter electrons more than the surrounding nucleoplasm 2292.

Photons 2280 are shown to emit from the nucleoli 2290 and are transmitted to the light detector 2288 (shown in Fig. 76), via light guide 2284. It is noted that the contrast obtained by detection of backscattered electrons and the contrast obtained by photon detection are due to different physical processes and to different chemical properties of features within the sample, and therefore do not generally overlap.

It is also noted that in accordance with a preferred embodiment of the present invention, imaging of the interior of the sample to a depth of up to approximately 2 microns is achievable when employing electron beams having an energy level of less than 50KeV, as seen in Fig. 77, wherein nucleoli 2290 disposed below electron beam permeable, fluid impermeable, membrane 2210 are imaged.

It is appreciated that the pre-microscopy multi-sample holder shown hereinabove in Figs. 21A-22B may be provided for use with SEM compatible sample containers of the type shown in Figs. 58A-77. Additionally, the pre-microscopy multi-sample holder may be associated with a suction device and pipettes shown hereinabove in Figs. 23A, 23B and 23C.

Reference is now made to Figs. 78A, 78B and 78C, which are simplified illustrations of a microscopy multi-sample holder in use with a SEM compatible sample dish of the type shown in Figs. 58A-67. As seen in Fig. 78A, the microscopy multi-sample holder preferably comprises a base 2400 and a sealing cover 2404. The base 2400 is preferably injection molded of a plastic material and defines an array of dish support locations 2406. Each dish support location 2406 is preferably defined by an aperture 2408 through which SEM microscopy may take place. Adjacent to each aperture 2408 there is preferably formed a pair of mutually aligned pairs of upstanding mutually spaced protrusions 2410 arranged to receive protrusions 2424 on sample dishes 2425. Sample dishes 2425 may be generally identical to sample dishes 2109, shown in Figs. 60A-62B, but do not require any threading or other attachment mechanism.

Base 2400 preferably also defines a plurality of liquid reservoirs 2412 which are adapted to hold liquid used to maintain a desired level of humidity in the interior of the microscopy multi-sample holder.

Sealing cover 2404 is preferably arranged for individual sealing engagement with each of sample dishes 2425. Preferably sealing cover 2404 is provided on the underside thereof with an array of 0-rings 2426, shown in Fig 78C, sealed thereto and arranged so as to sealingly engage a top rim surface of each of sample dishes 2425, when the sealing cover 2404 is in place, preferably in removable snap-fit engagement with base 2400.

Sealing cover 2404 is preferably provided with an array of light guides 2430 arranged to receive light from a sample in sample dish 2425 during light detection.

Light guides 2430 are inserted in apertures 2432 formed on sealing cover 2404.

Fig. 78B shows the apparatus of Fig. 78A with one sample dish 2425 positioned at a dish support location 2406 in base 2400. Fig. 78C shows sealing cover 2404 in snap fit engagement with base 2400, thereby providing individual sealing of each of sample dishes 2425 by means of O-ring 2426 and a portion of sealing cover 2404 circumscribed thereby.

Reference is now made to Figs. 79A, 79B and 79C, which are simplified illustrations of a microscopy multi-sample holder in use with a SEM compatible sample dish of the type shown in Figs. 68A-77. As seen in Fig. 79A, the microscopy multi-sample holder preferably comprises a base 2450 and a sealing cover 2454. The base 2450 is preferably injection molded of a plastic material and defines an array of dish support locations 2456. Each dish support location 2456 is preferably defined by an aperture 2458 through which SEM microscopy may take place. Adjacent to each aperture 2458 there is preferably formed a pair of mutually aligned pairs of upstanding mutually spaced protrusions 2460 arranged to receive protrusions 2474 on sample dishes 2475. Sample dishes 2475 may be generally identical to sample dishes 2209, shown in Figs. 70A-72B, but do not require any threading or other attachment mechanism.

Base 2450 preferably also defines a plurality of liquid reservoirs 2462 which are adapted to hold liquid used to maintain a desired level of humidity in the interior of the microscopy multi-sample holder.

Sealing cover 2454 is preferably arranged for individual sealing engagement of each of sample dishes 2475. Preferably sealing cover 2454 is provided on the underside thereof with an array of 0-rings 2476, shown in Fig 78C, sealed thereto and arranged so as to sealingly engage a top rim surface of each of sample dishes 2475, when the sealing cover 2454 is in place, preferably in removable snap-fit engagement with base 2450.

Preferably sealing cover 2454 is provided with an array of diaphragms 2477, shown in Fig. 79C, which may be identical to diaphragms 2218 described hereinabove with reference to Figs. 68A-77. Individual diaphragms 2477 are seated in a ring 2478 mounted over an aperture 2479 formed in sealing cover 2454.

Sealing cover 2454 is preferably provided with an array of light guides 2480 arranged to receive light from a sample in sample dish 2475 during light detection.

Light guides 2480 are inserted in apertures 2482 formed in sealing cover 2454.

Individual light guides 2480 are provided to collect light from a sample in sample dish 2475 during SEM inspection.

Fig. 79B shows the apparatus of Fig. 79A with one sample dish 2475 positioned at a dish support location 2456 in base 2450. Fig. 79C shows sealing cover 2454 in snap fit engagement with base 2450, thereby providing individual sealing of each of sample dishes 2475 by means of 0-ring 2476.

Reference is now made to Figs. 80A and 80B, which are simplified illustrations of a microscopy multi-sample holder defining a plurality of SEM compatible sample containers in accordance with a preferred embodiment of the present invention. As seen in Fig. 80A, the microscopy multi-sample holder preferably comprises a base 2500 and a sealing cover 2504. The base 2500 is preferably injection molded of a plastic material and defines an array of sample containers 2506. Each sample container 2506 preferably includes an aperture 2508 through which SEM microscopy may take place. An electron beam permeable, fluid impermeable, membrane 2510, shown in Fig. SOB, is sealed over each aperture 2508. Membrane 2510 is preferably identical to membrane 2110 described hereinabove with reference to Figs.

58A-67. Sealing cover 2504 preferably is arranged for individual sealing engagement with each of sample containers 2506.

Sealing cover 2504 is preferably provided with an array of light guides 2514 arranged to receive light from a sample in sample containers 2506 during light detection. Individual light guides 2514 are inserted into apertures 2516 formed in sealing cover 2504.

Fig. SOB shows the apparatus of Fig. 80A in sealed engagement, thereby providing individual sealing of each of sample containers 2506.

Reference is now made to Figs. 81A and 81B, which are simplified illustrations of a microscopy multi-sample holder defining a plurality of SEM compatible sample containers in accordance with a preferred embodiment of the present invention. As seen in Fig. 81A, the microscopy multi-sample holder preferably comprises a base 2550 and a sealing cover 2554. The base 2550 is preferably injection molded of a plastic material and defines an array of sample containers 2556. Each sample container 2556 preferably includes an aperture 2558 through which SEM microscopy may take place.

An electron beam permeable, fluid impermeable, membrane 2560, shown in Fig. 81B, is sealed over each aperture 2558. Membrane 2560 is preferably identical to membrane 2210 described hereinabove with reference to Figs. 68A-77.

Preferably sealing cover 2554 is provided with an array of diaphragms 2561, shown in Fig. 81B, which may be identical to diaphragms 2218 described hereinabove with reference to Figs. 68A-77. Individual diaphragms 2561 are seated in a ring 2562 mounted over an aperture 2563 formed in sealing cover 2554.

Sealing cover 2554 is preferably provided with an array of light guides 2564 arranged to receive from a sample in sample containers 2556 during light detection. Individual light guides 2564 are inserted in apertures 2566 formed in sealing cover 2554.

Fig. 81B shows the apparatus of Fig. 81A in sealed engagement, thereby providing individual sealing of each of sample containers 2556.

Reference is now made to Fig. 82, which is a simplified illustration of a SEM based sample inspection system and a light detection system constructed and operative in accordance with a preferred embodiment of the present invention. As seen in Fig. 82, a plurality of pre-microscopy multi-sample holders 2600, each containing a multiplicity of SEM compatible sample containers 2602 of the type shown in Figs. 58A

- 77, is shown in an incubator 2604. Preferably, light microscopy inspection of the samples in containers 2602 is carried out while the containers 2602 are mounted in holder 2600, as indicated at reference numeral 2606, in order to identify samples of interest. Preferably an inverted light microscope 2608 is employed for this purpose.

Preferably automated positioning systems, such as robotic arms, as shown, are used for conveying the pre-microscopy multi-sample holders 2600 and the containers 2602 throughout the system, it being appreciated that manual intervention may be employed at one or more stages as appropriate.

Thereafter, individual containers 2602 are removed from holders 2600 and placed on a removable electron microscope specimen stage 2610, which is subsequently introduced into a scanning electron microscope 2612.

The resulting image from the SEM inspection may be inspected visually by an operator and/or analyzed by conventional image analysis functionality, typically embodied in a computer 2614.

Preferably, light inspection of the samples in containers 2602 is carried out while the containers 2602 are in the SEM 2612. Preferably a light guide 2620 is employed to receive light from a light guide (not shown) inserted in container 2602 and to transmit the light to a light detector, such as a PMT 2622.

The resulting image from the light inspection may be inspected visually by an operator and/or analyzed by conventional image analysis functionality, typically embodied in a computer 2624.

Reference is now made to Fig. 83, which is a simplified illustration of a SEM based sample inspection system and a light detection system constructed and operative in accordance with another preferred embodiment of the present invention. As seen in Fig. 83, a plurality of microscopy multi-sample holders 2650, each containing a multiplicity of SEM compatible sample dishes 2652 of either of the types shown in Figs. 78A-79C, is shown in an incubator 2654. Preferably, light microscopy inspection of the samples in sample dishes 2652 is carried out while the sample dishes are mounted in holder 2650, as indicated at reference numeral 2656, in order to identify samples of interest. Preferably an inverted light microscope 2658 is employed for this purpose.

Preferably automated positioning systems, such as robotic arms, as shown, are used for conveying the microscopy multi-sample holders 2650 containing

sample dishes 2652 throughout the system, it being appreciated that manual intervention may be employed at one or more stages as appropriate.

Thereafter, holders 2650 are placed on an electron microscope specimen stage 2660, which is subsequently introduced into a scanning electron microscope 2662.

The resulting images may be inspected visually by an operator and/or analyzed by conventional image analysis functionality, typically embodied in a computer 2664.

Preferably, light inspection of the samples in sample dishes 2652 is carried out while the sample dishes 2652 are in the SEM 2662. Preferably a light guide 2666 is employed to receive light from a light guide (not shown) inserted in sample dishes 2652 and to transmit the light to a light detector, such as a PMT 2668.

The resulting image from the light inspection may be inspected visually by an operator and/or analyzed by conventional image analysis functionality, typically embodied in a computer 2669.

Reference is now made to Fig. 84, which is a simplified illustration of a SEM based sample inspection system and a light detection system constructed and operative in accordance with yet another preferred embodiment of the present invention.

As seen in Fig. 84, a plurality of microscopy multi-sample holders 2670, each defining a multiplicity of SEM compatible sample containers 2672, as shown in any of Figs. 80A- 81B, is seen in an incubator 2674. Preferably, light microscopy inspection of the samples in sample containers 2672 is carried out holder-wise, as indicated at reference numeral 2676, preferably in order to identify samples of interest. Preferably an inverted light microscope 2678 is employed for this purpose.

Preferably automated positioning systems, such as robotic arms, as shown, are used for conveying the microscopy multi-sample holders 2670 throughout the system, it being appreciated that manual intervention may be employed at one or more stages as appropriate.

Thereafter, holders 2670 are placed on an electron microscope specimen stage 2680, which is subsequently introduced into a scanning electron microscope 2682.

The resulting images may be inspected visually by an operator and/or analyzed by conventional image analysis functionality, typically embodied in a computer 2684.

Preferably, light inspection of the samples in containers 2672 is carried out while the containers 2672 are in the SEM 2682. Preferably a light guide 2686 is employed to receive light from a light guide (not shown) inserted in containers 2672 and to transmit the light to a light detector, such as a PMT 2688.

The resulting image from the light inspection may be inspected visually by an operator and/or analyzed by conventional image analysis functionality, typically embodied in a computer 2689.

Reference is made to Fig. 85, which is a simplified partially pictorial and partially sectional illustration of SEM inspection of a sample constructed and operative in accordance with another preferred embodiment of the present invention. As seen in Fig. 85, a sample container 3000 containing a sample 3002 is seated in a generally central aperture 3004 formed in a roof 3006 of an electron gun assembly 3010 and is positioned and sized so as to allow impingement of a focused electron beam 3012 on sample 3002 during SEM inspection.

Container 3000 is seated over an O-ring 3014 located in an interior surface 3016 of roof 3006. Container 3000 includes an electron-permeable, fluid impermeable, membrane 3020 adhered to the underside of a peripheral ring 3022 of sample container 3000. Sample 3002 lies over electron beam permeable, fluid impermeable membrane 3020.

Electron gun assembly 3010, which is part of a SEM inspection assembly, is provided with an electron gun (not shown) operative to provide electron beam 3012 emitted through a pole piece 3024 in a generally upward direction.

The electron beam 3012, generated by the electron gun, is shown to travel along a path 3026. The electron beam 3012 passes through electron beam permeable, fluid impermeable membrane 3020 and impinges on sample 3002 within sample container 3000. Electrons backscatter from sample 3002 and pass back through electron beam permeable, fluid impermeable membrane 3020 and are preferably detected by a backscattered electron detector 3030 in the electron gun assembly 3010.

It is a particular feature of the present invention that the electron beam 3020 impinges on sample 3002 on the underside thereof, thereby inspecting a sample region lying against electron beam permeable, fluid impermeable membrane 3020.

The electron gun assembly 3010 defines an electron gun assembly internal volume 3032. Internal volume 3032 is sealed by walls of the electron gun assembly 3010 and the container 3000 so as to maintain an evacuated environment within internal volume 3032, typically at a vacuum of 10-2-10-6 millibars, during SEM inspection.

Electron gun assembly 3010 also preferably includes a safety valve system comprising an airlock 3040 and a top wall 3042 defining a safety valve system internal volume 3044. Prior to removal of container 3000 the airlock 3040 is locked, so as to maintain an evacuated environment within internal volume 3044, typically at a vacuum of 10-z-10'6 millibars, during container removal. After the airlock 3040 is locked, a gas, typically nitrogen, is introduced via inlet tube 3046 into internal volume 3032 of electron gun assembly 3010. Container 3000 is then removed and preferably replaced. Alternatively, another container 3000 may then be placed in sample dish assembly 3010. After container 3000 or another container is introduced into the electron gun assembly 3010, the gas is pumped out, typically through outlet tube 3048, by a pump (not shown) and airlock 3040 is unlocked.

Airlock 3040 also preferably is operative to rapidly isolate internal volume 3044 from fluid that may enter the electron gun assembly 3010 from the ambient, due to leakage through the sample container 3000 or aperture 3004, or through lesions in electron beam permeable, fluid impermeable membrane 3020.

In accordance with another preferred embodiment of the present invention, electron gun assembly 3010 includes an adjustable seat for sample container 3000. The adjustable seat allows high-magnification imaging of different regions within sample 3002.

Reference is made to Fig. 86A-87B, which are simplified partially pictorial and partially sectional illustrations of a tissue sample slicing assembly constructed and operative in accordance with a preferred embodiment of the present invention. As seen in Fig. 86A, a plurality of stacked removable rectangular shaped slabs 3050 are mounted on a stage 3052. Preferably, protrusions 3054 are provided to retain slabs 3050 on stage 3052. A generally central aperture 3056 is formed in each individual slab 3050 and defines part of a recess 3058. A conventional slicing instrument 3060, such as a razor blade 3062, is preferably provided and is operative to

slice a tissue sample 3064 seated in recess 3058. The tissue sample 3064 is shown in Fig. 86A to extend beyond the top of the stack of slabs 3050.

Fig. 86B shows a plurality of slabs 3050, including a top slab 3066, providing a tissue sample thickness of XI. The slicing instrument 3060 slices the tissue sample 3064 into two portions, top portion 3068 and bottom portion 3070, where resulting bottom portion 3070 has a thickness of XI.

Fig. 87A illustrates the tissue sample slicing assembly of Fig. 86A with a plurality of slabs 3070, providing a tissue sample thickness of X2, where X2 < XI of Figs. 86A and 86B. As seen in Fig. 87B, the slicing instrument 3060 slices the tissue sample 3064 into two portions, top portion 3074 and bottom portion 3076, where resulting bottom portion 3076 has a thickness of X2.

It will be appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and described herein above. Rather the scope of the present invention includes both combinations and subcombinations of the various features described hereinabove as well as variations and modifications which would occur to persons skilled in the art upon reading the specifications and which are not in the prior art.